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    Structured Review

    Thermo Fisher cdna synthesis
    Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical <t>cDNA</t> replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.
    Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27019 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

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

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

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00096

    Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.
    Figure Legend Snippet: Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.

    Techniques Used: Expressing, Construct

    2) Product Images from "Transforming growth factor β-induced epithelial to mesenchymal transition requires the Ste20-like kinase SLK independently of its catalytic activity"

    Article Title: Transforming growth factor β-induced epithelial to mesenchymal transition requires the Ste20-like kinase SLK independently of its catalytic activity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21928

    SLK knockdown significantly inhibits Snai1 and vimentin expression following TGFβ1 treatment (A) NMuMG cells were infected with either AdshScrambled or AdshSLK for 48 hours. The cultures were stimulated with TGFβ and with and surveyed for SLK and vimentin expression. (B) Total RNA was extracted from identical cultures and Snai1 (B) and E-Cadherin (C) mRNA levels were monitored by Q-PCR. Normalization was performed against GAPDH mRNA levels. Each experiment was run in triplicate with three biological replicates. * p
    Figure Legend Snippet: SLK knockdown significantly inhibits Snai1 and vimentin expression following TGFβ1 treatment (A) NMuMG cells were infected with either AdshScrambled or AdshSLK for 48 hours. The cultures were stimulated with TGFβ and with and surveyed for SLK and vimentin expression. (B) Total RNA was extracted from identical cultures and Snai1 (B) and E-Cadherin (C) mRNA levels were monitored by Q-PCR. Normalization was performed against GAPDH mRNA levels. Each experiment was run in triplicate with three biological replicates. * p

    Techniques Used: Expressing, Infection, Polymerase Chain Reaction

    SLK regulates EMT independently of its kinase activity (A) NMuMG cells were treated with 2ng/mL of TGFβ1 for various times and SLK was immunoprecipitated and subjected to in vitro kinase assays. IP= immunoprecipitate, IB= immunoblot, WCL= whole cell lysate. (B) NMuMG cells were transfected with a wildtype or dominant negative (K63R) SLK construct and total SLK was immunoprecipitated and assayed for kinase activity. IB= immunoblot. Total RNA was also extracted from the transfected cultures following TGFβ stimulation (2ng/ml for 9 hours) and assayed for SLK (C) or Snai1 (D) expression. mRNA levels were normalized to GAPDH and directly compared to AdshSLK-infected cultures. Each experiment was run in triplicate with three biological replicates. Error bars represent the standard error. * p
    Figure Legend Snippet: SLK regulates EMT independently of its kinase activity (A) NMuMG cells were treated with 2ng/mL of TGFβ1 for various times and SLK was immunoprecipitated and subjected to in vitro kinase assays. IP= immunoprecipitate, IB= immunoblot, WCL= whole cell lysate. (B) NMuMG cells were transfected with a wildtype or dominant negative (K63R) SLK construct and total SLK was immunoprecipitated and assayed for kinase activity. IB= immunoblot. Total RNA was also extracted from the transfected cultures following TGFβ stimulation (2ng/ml for 9 hours) and assayed for SLK (C) or Snai1 (D) expression. mRNA levels were normalized to GAPDH and directly compared to AdshSLK-infected cultures. Each experiment was run in triplicate with three biological replicates. Error bars represent the standard error. * p

    Techniques Used: Activity Assay, Immunoprecipitation, In Vitro, Transfection, Dominant Negative Mutation, Construct, Expressing, Infection

    3) Product Images from "Comparative effects of the herbal constituent parthenolide (Feverfew) on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver"

    Article Title: Comparative effects of the herbal constituent parthenolide (Feverfew) on lipopolysaccharide-induced inflammatory gene expression in murine spleen and liver

    Journal: Journal of Inflammation (London, England)

    doi: 10.1186/1476-9255-2-6

    TNF-α protein production in sera following parthenolide and LPS co-treatment. Mice were treated and sera analyzed for TNF-α as described in Fig. 1 legend. The letter (a) indicates a significant difference compared to vehicle and parthenolide controls. Data are mean ± SEM (n = 16, controls n = 4), and is a combination of 4 separate experiments.
    Figure Legend Snippet: TNF-α protein production in sera following parthenolide and LPS co-treatment. Mice were treated and sera analyzed for TNF-α as described in Fig. 1 legend. The letter (a) indicates a significant difference compared to vehicle and parthenolide controls. Data are mean ± SEM (n = 16, controls n = 4), and is a combination of 4 separate experiments.

    Techniques Used: Mouse Assay

    TNF-α mRNA expression levels in spleen and liver following parthenolide and LPS co-treatment. Mice were treated and analyzed for TNF-α mRNA described in Fig. 2 legend. TNF-α mRNA levels were normalized using 18S rRNA and related to spleen control values. Data are mean ± SEM (n = 16, controls n = 4), and is a combination of 4 separate experiments.
    Figure Legend Snippet: TNF-α mRNA expression levels in spleen and liver following parthenolide and LPS co-treatment. Mice were treated and analyzed for TNF-α mRNA described in Fig. 2 legend. TNF-α mRNA levels were normalized using 18S rRNA and related to spleen control values. Data are mean ± SEM (n = 16, controls n = 4), and is a combination of 4 separate experiments.

    Techniques Used: Expressing, Mouse Assay

    4) Product Images from "The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability"

    Article Title: The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.90642.2008

    MCP-1-induced actin cytoskeleton reorganization in podocytes. A : filamentous (F)-actin strands were visible as cytoplasmic stress fibers in control cells. B : exposure to MCP-1 decreased the density of stress fibers and increased the localization of actin to bundles at the cell periphery (arrows). C : TGF-β-treated cells also showed a loss of stress fibers and increased peripheral actin. D – F : changes in F-actin appearance described above were not affected by an irrelevant hamster IgG, an isotype control. G – I : neutralizing anti-MCP-1 antibody (hamster, 30 μg/ml) greatly reduced the arrangement of F-actin near the cell margins that was secondary to MCP-1 or TGF-β. J – L : RS102895 (6 μM), a specific CCR2 inhibitor, also effectively blocked the MCP-1- or TGF-β-induced actin cytoskeletal reorganization. Magnification: ×400.
    Figure Legend Snippet: MCP-1-induced actin cytoskeleton reorganization in podocytes. A : filamentous (F)-actin strands were visible as cytoplasmic stress fibers in control cells. B : exposure to MCP-1 decreased the density of stress fibers and increased the localization of actin to bundles at the cell periphery (arrows). C : TGF-β-treated cells also showed a loss of stress fibers and increased peripheral actin. D – F : changes in F-actin appearance described above were not affected by an irrelevant hamster IgG, an isotype control. G – I : neutralizing anti-MCP-1 antibody (hamster, 30 μg/ml) greatly reduced the arrangement of F-actin near the cell margins that was secondary to MCP-1 or TGF-β. J – L : RS102895 (6 μM), a specific CCR2 inhibitor, also effectively blocked the MCP-1- or TGF-β-induced actin cytoskeletal reorganization. Magnification: ×400.

    Techniques Used:

    MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A : in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 ( n = 4). * P
    Figure Legend Snippet: MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A : in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 ( n = 4). * P

    Techniques Used: Scratch Wound Assay Assay, Migration, Inhibition

    TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A : cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 ( n = 3). * P
    Figure Legend Snippet: TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A : cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 ( n = 3). * P

    Techniques Used: Expressing, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

    Proposed TGF-β-induced MCP-1/CCR2 loop in podocytes. 1 : The diabetic milieu stimulates mesangial cells to produce TGF-β. 2 : TGF-β binds to its type II receptor, which is upregulated in the podocytes in diabetes. 3 : Podocytes are stimulated to produce MCP-1 via TGF-β type I receptor signaling and the PI3K pathway. 4 : Via the CCR2 receptor, MCP-1 causes increased podocyte migration, actin cytoskeletal changes, and albumin hyperpermeability.
    Figure Legend Snippet: Proposed TGF-β-induced MCP-1/CCR2 loop in podocytes. 1 : The diabetic milieu stimulates mesangial cells to produce TGF-β. 2 : TGF-β binds to its type II receptor, which is upregulated in the podocytes in diabetes. 3 : Podocytes are stimulated to produce MCP-1 via TGF-β type I receptor signaling and the PI3K pathway. 4 : Via the CCR2 receptor, MCP-1 causes increased podocyte migration, actin cytoskeletal changes, and albumin hyperpermeability.

    Techniques Used: Migration

    Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A : a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes ( n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. * P
    Figure Legend Snippet: Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A : a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes ( n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. * P

    Techniques Used: Cell Culture

    Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A : CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining ( inset ). B : no fluorescence is detected when the primary antibody is omitted. C : staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D : staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E : RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.
    Figure Legend Snippet: Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A : CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining ( inset ). B : no fluorescence is detected when the primary antibody is omitted. C : staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D : staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E : RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.

    Techniques Used: Staining, Fluorescence, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Negative Control

    MCP-1 increases podocyte permeability to albumin. A : Evans blue-labeled albumin (EBA) can be quantified by the absorbance characteristics of the Evans blue dye, which absorbs light most strongly at 620 nm. In our tests, the A 620 measurement is oblivious to DMEM but is quite sensitive to the mixture of DMEM+EBA. B : in a Transwell setup to assay the cellular permeability to EBA, performed after the transepithelial electrical resistance had plateaued at 114.5 ± 24.6 Ω·cm 2 , significantly more EBA had diffused from the lower into the upper chamber across the podocyte monolayer at 24 h as a result of MCP-1 treatment (50 ng/ml) vs. control. TGF-β (2 ng/ml) had a similar effect on EBA transit, although not statistically significant. The permeability to albumin induced by MCP-1 or TGF-β was returned to control levels by concurrent treatment with RS102895 (6 μM), an inhibitor of CCR2 signaling ( n = 4). * P
    Figure Legend Snippet: MCP-1 increases podocyte permeability to albumin. A : Evans blue-labeled albumin (EBA) can be quantified by the absorbance characteristics of the Evans blue dye, which absorbs light most strongly at 620 nm. In our tests, the A 620 measurement is oblivious to DMEM but is quite sensitive to the mixture of DMEM+EBA. B : in a Transwell setup to assay the cellular permeability to EBA, performed after the transepithelial electrical resistance had plateaued at 114.5 ± 24.6 Ω·cm 2 , significantly more EBA had diffused from the lower into the upper chamber across the podocyte monolayer at 24 h as a result of MCP-1 treatment (50 ng/ml) vs. control. TGF-β (2 ng/ml) had a similar effect on EBA transit, although not statistically significant. The permeability to albumin induced by MCP-1 or TGF-β was returned to control levels by concurrent treatment with RS102895 (6 μM), an inhibitor of CCR2 signaling ( n = 4). * P

    Techniques Used: Permeability, Labeling

    5) Product Images from "A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin"

    Article Title: A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.13.4667-4676.2002

    Expression of cLys domain genes in mouse macrophages. (A) cLys expression was determined by use of Competimer-normalized RT-PCR, as described in Materials and Methods. 18S mouse rRNA was used as an internal standard. Lanes 2 to 4 show results from female macrophages subjected to no selection (NS) and to 6-TG and HAT selection, respectively. Lanes 5 and 6 show male macrophages subjected to no selection and 6-TG selection, respectively. Lanes 7 to 9 show female cells identical to those in lanes 2 to 4 except for the addition of LPS treatment. Similarly, lanes 10 and 11 are male cells identical to those in lanes 5 and 6 except for the addition of LPS. Lanes 1 and 12 are control reaction mixtures containing a single set of PCR primers for either cLys (lane 1) or 18S rRNA (lane 12) only. Lane 13 is a no-template control. (B) cGas41 expression in mouse macrophages. 18S mouse rRNA was used as an internal standard. Lane M shows size markers.
    Figure Legend Snippet: Expression of cLys domain genes in mouse macrophages. (A) cLys expression was determined by use of Competimer-normalized RT-PCR, as described in Materials and Methods. 18S mouse rRNA was used as an internal standard. Lanes 2 to 4 show results from female macrophages subjected to no selection (NS) and to 6-TG and HAT selection, respectively. Lanes 5 and 6 show male macrophages subjected to no selection and 6-TG selection, respectively. Lanes 7 to 9 show female cells identical to those in lanes 2 to 4 except for the addition of LPS treatment. Similarly, lanes 10 and 11 are male cells identical to those in lanes 5 and 6 except for the addition of LPS. Lanes 1 and 12 are control reaction mixtures containing a single set of PCR primers for either cLys (lane 1) or 18S rRNA (lane 12) only. Lane 13 is a no-template control. (B) cGas41 expression in mouse macrophages. 18S mouse rRNA was used as an internal standard. Lane M shows size markers.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Selection, HAT Assay, Polymerase Chain Reaction

    6) Product Images from "miR-202-3p Regulates Sertoli Cell Proliferation, Synthesis Function, and Apoptosis by Targeting LRP6 and Cyclin D1 of Wnt/β-Catenin Signaling"

    Article Title: miR-202-3p Regulates Sertoli Cell Proliferation, Synthesis Function, and Apoptosis by Targeting LRP6 and Cyclin D1 of Wnt/β-Catenin Signaling

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.10.012

    Isolation and Identification of Human Sertoli Cells from OA and SCOS Patients (A) RT-PCR showed the transcripts of GDNF , GATA 4, S OX9 , WT1 , BMP4, and SCF in the isolated cells. PCR with PBS but without cDNA served as a negative control. (B) Western blots showed the protein levels of BMP4, SCF, and GDNF in OA and SCOS Sertoli cells. (C–L) Immunofluorescence demonstrated the expression of SOX9 (C), GATA4 (D), WT1 (E), VIM (F), GDNF (G), OCLN (H), SCF (I), VASA (J), α-SMA (K), and CYP11A1 (L) in the isolated cells. Replacement of primary antibodies with PBS was used as a negative control (M). The cell nuclei were stained with DAPI. Scale bars, 5 μm (C–M).
    Figure Legend Snippet: Isolation and Identification of Human Sertoli Cells from OA and SCOS Patients (A) RT-PCR showed the transcripts of GDNF , GATA 4, S OX9 , WT1 , BMP4, and SCF in the isolated cells. PCR with PBS but without cDNA served as a negative control. (B) Western blots showed the protein levels of BMP4, SCF, and GDNF in OA and SCOS Sertoli cells. (C–L) Immunofluorescence demonstrated the expression of SOX9 (C), GATA4 (D), WT1 (E), VIM (F), GDNF (G), OCLN (H), SCF (I), VASA (J), α-SMA (K), and CYP11A1 (L) in the isolated cells. Replacement of primary antibodies with PBS was used as a negative control (M). The cell nuclei were stained with DAPI. Scale bars, 5 μm (C–M).

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control, Western Blot, Immunofluorescence, Expressing, Staining

    7) Product Images from "In Vitro and In Vivo Analysis of the Interaction between RNA Helicase A and HIV-1 RNA"

    Article Title: In Vitro and In Vivo Analysis of the Interaction between RNA Helicase A and HIV-1 RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.01993-12

    RHA binding to viral RNA in vivo using an RNA-protein coprecipitation assay. 293T cells were cotransfected with a BH10 vector containing full-length HIV-1 BH10 proviral DNA and a plasmid expressing His-tagged RHA or only the 6×His tag. Twenty-four
    Figure Legend Snippet: RHA binding to viral RNA in vivo using an RNA-protein coprecipitation assay. 293T cells were cotransfected with a BH10 vector containing full-length HIV-1 BH10 proviral DNA and a plasmid expressing His-tagged RHA or only the 6×His tag. Twenty-four

    Techniques Used: Binding Assay, In Vivo, Plasmid Preparation, Expressing

    Ability of RHA to bind to different regions of HIV-1 genomic RNA. (A) Native gel electrophoresis of synthetic HIV-1 RNA fragments. The HIV-1 RNAs were synthesized by in vitro transcription, labeled with 32 pCp, separated in native 6% polyacrylamide gels,
    Figure Legend Snippet: Ability of RHA to bind to different regions of HIV-1 genomic RNA. (A) Native gel electrophoresis of synthetic HIV-1 RNA fragments. The HIV-1 RNAs were synthesized by in vitro transcription, labeled with 32 pCp, separated in native 6% polyacrylamide gels,

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, In Vitro, Labeling

    Role of dsRBDs in the in vivo promotion of tRNA 3 Lys annealing to HIV-1 RNA by RHA. 293T cells were first transfected with siRNA Con or siRNA RHA and were then cotransfected 16 h later with a BH10 vector coding for HIV-1 BH10 and a plasmid expressing either
    Figure Legend Snippet: Role of dsRBDs in the in vivo promotion of tRNA 3 Lys annealing to HIV-1 RNA by RHA. 293T cells were first transfected with siRNA Con or siRNA RHA and were then cotransfected 16 h later with a BH10 vector coding for HIV-1 BH10 and a plasmid expressing either

    Techniques Used: In Vivo, Transfection, Plasmid Preparation, Expressing

    8) Product Images from "A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression"

    Article Title: A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddu352

    The MTPAP 1432A > G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. ( A ) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C , unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A 0 ) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A 50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. ( B ) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. ( C ) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. ( D ) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t -test (ns * P
    Figure Legend Snippet: The MTPAP 1432A > G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. ( A ) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C , unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A 0 ) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A 50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. ( B ) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. ( C ) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. ( D ) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t -test (ns * P

    Techniques Used: Mutagenesis, Isolation, Transduction, Migration, SDS Page, Imaging, Blue Native PAGE

    In vitro polyadenylation activity of mtPAP. ( A ) Polyadenylation activity of recombinant wild-type (WT; lanes 2–4) and mutant (p.N478D; lanes 6–8) mitochondrial poly(A) polymerase (0.55 µ m ) was determined with increasing ATP concentrations. The RNA substrate was an unadenylated 277-nt 3′ fragment of MTND3 (0.25 µ m ). The right hand panel contains an IVT RNA artefact (500 nt) present in the absence of mtPAP (lane 5). Reactions were quenched with 90% formamide/1× TBE, separated through a 6% polyacrylamide/8.3 m urea gel, then stained with SYBR gold and visualized by scanning with a Typhoon FLA 9500 instrument. ( B ) Short RNAs (0.25 µ m ) corresponding to the final 40 nucleotides of RNA14 with (A8, lanes 5–8) or without (A0, lanes 1–4) an oligo(A8) addition were used as templates for polyadenylation by recombinant wild-type (WT; lanes 2 and 6) or mutant (p.N478D; lanes 3 and 7) mtPAP (0.55 µ m ). An equal amount of BSA was added in a parallel experiment as a control (lanes 4 and 8). Products were separated through 15% polyacrylamide/8.3 m urea gel and visualized as in (A). ( C ) Increasing amounts of wild-type mtPAP (34 n m to 0.55 µ m ) were added to the short RNA14 A0 (0.25 µ m ) template in the presence (lanes 8–12) or absence (lanes 2–6) of LRPRRC/SLIRP complex. A higher molecular species (*) of varying intensity was observed with wild-type mtPAP. Products were separated and visualized as in (B).
    Figure Legend Snippet: In vitro polyadenylation activity of mtPAP. ( A ) Polyadenylation activity of recombinant wild-type (WT; lanes 2–4) and mutant (p.N478D; lanes 6–8) mitochondrial poly(A) polymerase (0.55 µ m ) was determined with increasing ATP concentrations. The RNA substrate was an unadenylated 277-nt 3′ fragment of MTND3 (0.25 µ m ). The right hand panel contains an IVT RNA artefact (500 nt) present in the absence of mtPAP (lane 5). Reactions were quenched with 90% formamide/1× TBE, separated through a 6% polyacrylamide/8.3 m urea gel, then stained with SYBR gold and visualized by scanning with a Typhoon FLA 9500 instrument. ( B ) Short RNAs (0.25 µ m ) corresponding to the final 40 nucleotides of RNA14 with (A8, lanes 5–8) or without (A0, lanes 1–4) an oligo(A8) addition were used as templates for polyadenylation by recombinant wild-type (WT; lanes 2 and 6) or mutant (p.N478D; lanes 3 and 7) mtPAP (0.55 µ m ). An equal amount of BSA was added in a parallel experiment as a control (lanes 4 and 8). Products were separated through 15% polyacrylamide/8.3 m urea gel and visualized as in (A). ( C ) Increasing amounts of wild-type mtPAP (34 n m to 0.55 µ m ) were added to the short RNA14 A0 (0.25 µ m ) template in the presence (lanes 8–12) or absence (lanes 2–6) of LRPRRC/SLIRP complex. A higher molecular species (*) of varying intensity was observed with wild-type mtPAP. Products were separated and visualized as in (B).

    Techniques Used: In Vitro, Activity Assay, Recombinant, Mutagenesis, Staining

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

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

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00096

    Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.
    Figure Legend Snippet: Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.

    Techniques Used: Expressing, Construct

    10) Product Images from "Inhibition of JNK by compound C66 prevents pathological changes of the aorta in STZ-induced diabetes"

    Article Title: Inhibition of JNK by compound C66 prevents pathological changes of the aorta in STZ-induced diabetes

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12267

    Protective effect of C66 on diabetes-induced aortic tumour necrosis factor-alpha (TNF-α) expression. Aortic expression of TNF-α was examined by immunohistochemical staining for its protein (A) expression in aortic tunica media, followed by semi-quantitative analysis (B) and real-time PCR for its mRNA level (C). Data were presented as means ± SD ( n = 5). * P
    Figure Legend Snippet: Protective effect of C66 on diabetes-induced aortic tumour necrosis factor-alpha (TNF-α) expression. Aortic expression of TNF-α was examined by immunohistochemical staining for its protein (A) expression in aortic tunica media, followed by semi-quantitative analysis (B) and real-time PCR for its mRNA level (C). Data were presented as means ± SD ( n = 5). * P

    Techniques Used: Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction

    11) Product Images from "The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory"

    Article Title: The brain-tumor related protein podoplanin regulates synaptic plasticity and hippocampus-dependent learning and memory

    Journal: Annals of Medicine

    doi: 10.1080/07853890.2016.1219455

    Podoplanin deletion hampers NGF-induced neuritic outgrowth. (a) Hippocampal neurons from wild-type mice developed observable neuritic extensions after three days in regular culture conditions (left). Neurites appear lengthened after three days of daily treatment with 50 ng/mL of NGF (middle). Right, bar graph comparison shows significant increase induced by NGF. (b) Hippocampal neurons from podoplanin−/− mice developed neuritic extensions comparable to those of wild-type after three days in regular conditions (left) but NGF treatment did not enhance neurite length (middle and right bar graph). Data are displayed as mean ± SEM. **(p ≤ 0.01), ns (p > 0.05).
    Figure Legend Snippet: Podoplanin deletion hampers NGF-induced neuritic outgrowth. (a) Hippocampal neurons from wild-type mice developed observable neuritic extensions after three days in regular culture conditions (left). Neurites appear lengthened after three days of daily treatment with 50 ng/mL of NGF (middle). Right, bar graph comparison shows significant increase induced by NGF. (b) Hippocampal neurons from podoplanin−/− mice developed neuritic extensions comparable to those of wild-type after three days in regular conditions (left) but NGF treatment did not enhance neurite length (middle and right bar graph). Data are displayed as mean ± SEM. **(p ≤ 0.01), ns (p > 0.05).

    Techniques Used: Mouse Assay

    A proposed model for podoplanin signaling in the brain neurons. Extracellular NGF (1) binds to LRR or Ig-like domains of its canonical TrkA receptor (2) (it can also bind to p75 receptors not represented here) to induce well established CREB-mediated neuritic outgrowth important for synaptic plasticity and learning and memory. Concomitantly, extracellular NGF (1) can physically interact with podoplanin (3) to modulate podoplanin-related signaling via intracellular partners like Ezrin (4) to promote cytoskeleton reorganization and hence neuritic protrusion or retraction thus affecting the synaptic function. Whether podoplanin can also directly interact with TrkA receptors (“?” in the figure) thus affecting NGF/TrkA-mediated CREB signaling or whether podoplanin may, on its own ability, affect NGF-mediated CREB signaling (5) via a NGF/podoplanin signaling pathway (“??” in the figure) remains to be characterized.
    Figure Legend Snippet: A proposed model for podoplanin signaling in the brain neurons. Extracellular NGF (1) binds to LRR or Ig-like domains of its canonical TrkA receptor (2) (it can also bind to p75 receptors not represented here) to induce well established CREB-mediated neuritic outgrowth important for synaptic plasticity and learning and memory. Concomitantly, extracellular NGF (1) can physically interact with podoplanin (3) to modulate podoplanin-related signaling via intracellular partners like Ezrin (4) to promote cytoskeleton reorganization and hence neuritic protrusion or retraction thus affecting the synaptic function. Whether podoplanin can also directly interact with TrkA receptors (“?” in the figure) thus affecting NGF/TrkA-mediated CREB signaling or whether podoplanin may, on its own ability, affect NGF-mediated CREB signaling (5) via a NGF/podoplanin signaling pathway (“??” in the figure) remains to be characterized.

    Techniques Used:

    Podoplanin: a potential new player in the NGF/TrkA signaling pathway. (a) Real-time monitoring for potential human podoplanin and human NGF macromolecular interactions using SPR data obtained in a BiaCore apparatus showed a relatively high-affinity binding of recombinant NGF to immobilized podoplanin protein, as expressed in relative binding units on the y axis vs time. No binding was observed when a control GDF-8 recombinant protein was used even at concentrations five times higher than those used for the NGF assay. A heparin-based solution was used as a dissociation buffer. (b) No differences in the basal levels of total Ezrin or p-Ezrin (c) were observed between wild-type and podoplanin−/− mice hippocampal neurons. However, primary-cultured hippocampal neurons presented with comparatively lower levels of p-Ezrin upon NGF stimulation (50 ng/mL, 20 min) (d). Comparably, no differences in the levels of TrkA were observed under basal untreated conditions (e) but reduced levels were found in neurons from podoplanin−/− after NGF treatment (50 ng/mL, 2 h) (f). (g) Levels of CREB were also enhanced upon NGF stimulation in neurons from wild-type mice whereas CREB levels did not alter in response to NGF treatment in podoplanin−/− cultured neurons. Data are displayed as mean ± SEM. ns (p > 0.05), *(p ≤ 0.05), **(p ≤ 0.01), ***(p ≤ 0.001).
    Figure Legend Snippet: Podoplanin: a potential new player in the NGF/TrkA signaling pathway. (a) Real-time monitoring for potential human podoplanin and human NGF macromolecular interactions using SPR data obtained in a BiaCore apparatus showed a relatively high-affinity binding of recombinant NGF to immobilized podoplanin protein, as expressed in relative binding units on the y axis vs time. No binding was observed when a control GDF-8 recombinant protein was used even at concentrations five times higher than those used for the NGF assay. A heparin-based solution was used as a dissociation buffer. (b) No differences in the basal levels of total Ezrin or p-Ezrin (c) were observed between wild-type and podoplanin−/− mice hippocampal neurons. However, primary-cultured hippocampal neurons presented with comparatively lower levels of p-Ezrin upon NGF stimulation (50 ng/mL, 20 min) (d). Comparably, no differences in the levels of TrkA were observed under basal untreated conditions (e) but reduced levels were found in neurons from podoplanin−/− after NGF treatment (50 ng/mL, 2 h) (f). (g) Levels of CREB were also enhanced upon NGF stimulation in neurons from wild-type mice whereas CREB levels did not alter in response to NGF treatment in podoplanin−/− cultured neurons. Data are displayed as mean ± SEM. ns (p > 0.05), *(p ≤ 0.05), **(p ≤ 0.01), ***(p ≤ 0.001).

    Techniques Used: SPR Assay, Binding Assay, Recombinant, Mouse Assay, Cell Culture

    Podoplanin is expressed in the mouse brain. (a) qPCR examinations in adult (8–10 weeks old) wild-type mouse tissues (n = 3 animals) indicated a salient presence of podoplanin in the brain. Transcription levels from triplicate experiments were normalized to values obtained from podoplanin in the heart, for descriptive proposes, as indicated by an asterisk. He = heart, Lu = lung, Li = liver, Sp = spleen, Ki = kidney, AT = adipose tissue, SM = skeletal muscle, Th = thymus, Pr = prostate, Br = total brain. (b) In a different experiment, qPCR analysis further verified the presence of podoplanin mRNAs in different areas across the brain relative to the hippocampus of 8–10 weeks old wild-type mice (n = 3). (c) Immunohistochemical staining of brain sections obtained from adult wild type mice showed a clear signal for podoplanin (upper) and no signal in negative controls where primary antibody was omitted (lower). Objective magnifications -from left to right- are 4×, 20×, 20×, and 40× for upper and lower pictures. Consistent results were found when the podoplanin antibody was used on brain sections obtained from the podoplanin knock out (Pdpn−/−) mice (lower pictures). (d) Western blot assay exclusively using hippocampal tissue from wild-type mice corroborated the expression of podoplanin. No signal was detected in hippocampal tissue from podoplanin−/− mice (right). Histograms data are displayed as mean ± SEM.
    Figure Legend Snippet: Podoplanin is expressed in the mouse brain. (a) qPCR examinations in adult (8–10 weeks old) wild-type mouse tissues (n = 3 animals) indicated a salient presence of podoplanin in the brain. Transcription levels from triplicate experiments were normalized to values obtained from podoplanin in the heart, for descriptive proposes, as indicated by an asterisk. He = heart, Lu = lung, Li = liver, Sp = spleen, Ki = kidney, AT = adipose tissue, SM = skeletal muscle, Th = thymus, Pr = prostate, Br = total brain. (b) In a different experiment, qPCR analysis further verified the presence of podoplanin mRNAs in different areas across the brain relative to the hippocampus of 8–10 weeks old wild-type mice (n = 3). (c) Immunohistochemical staining of brain sections obtained from adult wild type mice showed a clear signal for podoplanin (upper) and no signal in negative controls where primary antibody was omitted (lower). Objective magnifications -from left to right- are 4×, 20×, 20×, and 40× for upper and lower pictures. Consistent results were found when the podoplanin antibody was used on brain sections obtained from the podoplanin knock out (Pdpn−/−) mice (lower pictures). (d) Western blot assay exclusively using hippocampal tissue from wild-type mice corroborated the expression of podoplanin. No signal was detected in hippocampal tissue from podoplanin−/− mice (right). Histograms data are displayed as mean ± SEM.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Immunohistochemistry, Staining, Knock-Out, Western Blot, Expressing

    Podoplanin overexpression promotes synaptic activity and neuritic outgrowth. Hippocampal “sister” neurons from wild-type mice and cultured in identical conditions were transfected with a DNA plasmidic vector encoding for either the expression of the GFP alone (a) or with DNA encoding for a podoplanin-GFP fusion protein (b). Recordings of mEPSCs revealed a significant increase in the frequency of spontaneous synaptic events in neurons expressing podoplanin relative to those expressing GFP alone (representative current traces, upper panels a-b and bar graph, left in (c). Overexpressing of podoplanin also resulted in larger number of soma-derived neurites, which appeared shorter in length (pictures illustrating representative neurons, a-b lower panels, and bar graphs, middle and right in (c). Data are displayed as mean ± SEM. ***(p ≤ 0.001).
    Figure Legend Snippet: Podoplanin overexpression promotes synaptic activity and neuritic outgrowth. Hippocampal “sister” neurons from wild-type mice and cultured in identical conditions were transfected with a DNA plasmidic vector encoding for either the expression of the GFP alone (a) or with DNA encoding for a podoplanin-GFP fusion protein (b). Recordings of mEPSCs revealed a significant increase in the frequency of spontaneous synaptic events in neurons expressing podoplanin relative to those expressing GFP alone (representative current traces, upper panels a-b and bar graph, left in (c). Overexpressing of podoplanin also resulted in larger number of soma-derived neurites, which appeared shorter in length (pictures illustrating representative neurons, a-b lower panels, and bar graphs, middle and right in (c). Data are displayed as mean ± SEM. ***(p ≤ 0.001).

    Techniques Used: Over Expression, Activity Assay, Mouse Assay, Cell Culture, Transfection, Plasmid Preparation, Expressing, Derivative Assay

    Survivor podoplanin−/− mice don’t exhibit other distinctive phenotypic abnormalities. (a) Podoplanin−/− (Pdpn−/−) newborn mice (postnatal day PND1-3) presented without obvious differences in body (lower left) or brain (lower right) gross structural appearance compared to wild-type (WT) animals (upper left and right pictures, representative images from n = 4 animals per group analyzed). Small vessel morphology anomalies were observable in the cortex of some podoplanin−/− brains (arrow). (b) While body and brain morphology remained undistinguishable between wild-type and podoplanin−/− mice 10 days after birth (left pictures), vessel morphology anomalies were not any more visible in the brain of podoplanin−/− mice (right lower picture). (c) Also in mature adult mice, no apparent differences in body (left) and brain morphology (middle) and no vessel anomalies were observed for wild-type and podoplanin−/− mice (bars =1 cm). Stained brain coronal sections showed no obvious differences in shape and cytoarchitectonic organization for cortex, hippocampus or other structures for wild type and podoplanin−/− brains (right panels). (d) In adulthood, the levels of the synaptic proteins synaptophysin and synaptotagmin I (e), as well as physiological parameters including body weights (f) and body length (g) were also indistinguishable between wild-type and podoplanin−/− mice. ns (p > 0.05).
    Figure Legend Snippet: Survivor podoplanin−/− mice don’t exhibit other distinctive phenotypic abnormalities. (a) Podoplanin−/− (Pdpn−/−) newborn mice (postnatal day PND1-3) presented without obvious differences in body (lower left) or brain (lower right) gross structural appearance compared to wild-type (WT) animals (upper left and right pictures, representative images from n = 4 animals per group analyzed). Small vessel morphology anomalies were observable in the cortex of some podoplanin−/− brains (arrow). (b) While body and brain morphology remained undistinguishable between wild-type and podoplanin−/− mice 10 days after birth (left pictures), vessel morphology anomalies were not any more visible in the brain of podoplanin−/− mice (right lower picture). (c) Also in mature adult mice, no apparent differences in body (left) and brain morphology (middle) and no vessel anomalies were observed for wild-type and podoplanin−/− mice (bars =1 cm). Stained brain coronal sections showed no obvious differences in shape and cytoarchitectonic organization for cortex, hippocampus or other structures for wild type and podoplanin−/− brains (right panels). (d) In adulthood, the levels of the synaptic proteins synaptophysin and synaptotagmin I (e), as well as physiological parameters including body weights (f) and body length (g) were also indistinguishable between wild-type and podoplanin−/− mice. ns (p > 0.05).

    Techniques Used: Mouse Assay, Staining

    In vivo lack of podoplanin impairs hippocampus-dependent spatial learning and memory. (a) The latency to reach the hidden platform in the Morris water maze during three days of training (TD 1-3) is displayed as average of three trials per day. Escape latency was significantly higher in podoplanin−/− mice as compared to wild-type controls on TD 2 and TD 3. (b) During the probe trial, implemented to examine the retention in memory of the behavioral tasks, the percentage of time spent in the target quadrant (original location of the platform during training) was lower for podoplanin−/− animals compared to that of wild-type mice. No significant differences were observed either in the average speed of swimming (c) or in the performance on the Rota Rod test (d) between wild-type and podoplanin−/− mice. Data are displayed as mean ± SEM. **(p ≤ 0.01), ns (p > 0.05). (e) Podoplanin−/− mice exhibited a statistically significant reduction in the time of freezing when hippocampus-dependent contextual fear-conditioning was examined. (f) Podoplanin−/− mice present with amygdala-dependent cued fear conditioning indistinguishable from that from wild-type counterparts.
    Figure Legend Snippet: In vivo lack of podoplanin impairs hippocampus-dependent spatial learning and memory. (a) The latency to reach the hidden platform in the Morris water maze during three days of training (TD 1-3) is displayed as average of three trials per day. Escape latency was significantly higher in podoplanin−/− mice as compared to wild-type controls on TD 2 and TD 3. (b) During the probe trial, implemented to examine the retention in memory of the behavioral tasks, the percentage of time spent in the target quadrant (original location of the platform during training) was lower for podoplanin−/− animals compared to that of wild-type mice. No significant differences were observed either in the average speed of swimming (c) or in the performance on the Rota Rod test (d) between wild-type and podoplanin−/− mice. Data are displayed as mean ± SEM. **(p ≤ 0.01), ns (p > 0.05). (e) Podoplanin−/− mice exhibited a statistically significant reduction in the time of freezing when hippocampus-dependent contextual fear-conditioning was examined. (f) Podoplanin−/− mice present with amygdala-dependent cued fear conditioning indistinguishable from that from wild-type counterparts.

    Techniques Used: In Vivo, Mouse Assay

    Podoplanin deletion selectively impairs long-term synaptic plasticity in the hippocampal dentate gyrus. (a) Comparative examinations of LTP in CA3-Schaffer collateral-CA1 synapses (inset cartoon in left represents the positioning of stimulating and recording electrodes) of slices obtained from wild-type and podoplanin−/− mice (n = 9 animals per group). (b) No differences in basal synaptic transmission (left) was detected at the dentate gyrus (inset cartoon in left represents the positioning of stimulating and recording electrodes) in hippocampal slices from podoplanin−/− and control wild-type mice as examined by input/output protocols. Right, representative fEPSPs traces (upper insets); temporal courses (lower chart plot) of averaged field-postsynaptic-potential-slopes; and corresponding bar graphs of the end-time points (right) of data obtained before and after application of electrical stimulation inducing long-term-potentiation (LTP) in the dentate gyrus. A mixed-model repeated measure ANOVA indicated no main effect of genotype (p > 0.05), a highly significant main effect of time (F 158,82 = 57.05, p
    Figure Legend Snippet: Podoplanin deletion selectively impairs long-term synaptic plasticity in the hippocampal dentate gyrus. (a) Comparative examinations of LTP in CA3-Schaffer collateral-CA1 synapses (inset cartoon in left represents the positioning of stimulating and recording electrodes) of slices obtained from wild-type and podoplanin−/− mice (n = 9 animals per group). (b) No differences in basal synaptic transmission (left) was detected at the dentate gyrus (inset cartoon in left represents the positioning of stimulating and recording electrodes) in hippocampal slices from podoplanin−/− and control wild-type mice as examined by input/output protocols. Right, representative fEPSPs traces (upper insets); temporal courses (lower chart plot) of averaged field-postsynaptic-potential-slopes; and corresponding bar graphs of the end-time points (right) of data obtained before and after application of electrical stimulation inducing long-term-potentiation (LTP) in the dentate gyrus. A mixed-model repeated measure ANOVA indicated no main effect of genotype (p > 0.05), a highly significant main effect of time (F 158,82 = 57.05, p

    Techniques Used: Mouse Assay, Transmission Assay

    12) Product Images from "A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression"

    Article Title: A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddu352

    The LRPPRC/SLIRP complex modulates the polyadenylation activity of both wild-type and mutant mtPAP. ( A ) Polyadenylation activity was analysed using wild-type mtPAP (0.55 µ m ) incubated for 1h with the short RNA14 A0 substrate (0.25 µ m ), alone (lane 2), with LRPPRC (0.5 µ m lane 3; 1.25 µ m lane 6), LRPPRC/SLIRP complex (0.5 µ m , lane 4) or BSA (0.5 µ m , lane 5). Products were separated and visualized as described earlier. ( B ) The effect of LRPPRC/SLIRP complex (0.48 µ m ) on polyadenylation by wild-type mtPAP (0.55 µ m ) of an unadenylated (A0, lanes 2 and 3) compared with an oligoadenylated (A8, lanes 5 and 6) RNA14 substrate was analysed. Products were separated and visualized as described earlier. ( C ) The effect of LRPPRC/SLIRP complex (0.48 µ m ; lanes 3 and 5) on polyadenylation by p.N478D mutant (0.55 µ m ; lanes 4–5) poly(A) polymerase was compared with wild-type (0.55 µM; lanes 2–3) enzyme. The short unadenylated RNA14 A0 substrate (0.25 µ m ) was used. Products were separated and visualized as described earlier.
    Figure Legend Snippet: The LRPPRC/SLIRP complex modulates the polyadenylation activity of both wild-type and mutant mtPAP. ( A ) Polyadenylation activity was analysed using wild-type mtPAP (0.55 µ m ) incubated for 1h with the short RNA14 A0 substrate (0.25 µ m ), alone (lane 2), with LRPPRC (0.5 µ m lane 3; 1.25 µ m lane 6), LRPPRC/SLIRP complex (0.5 µ m , lane 4) or BSA (0.5 µ m , lane 5). Products were separated and visualized as described earlier. ( B ) The effect of LRPPRC/SLIRP complex (0.48 µ m ) on polyadenylation by wild-type mtPAP (0.55 µ m ) of an unadenylated (A0, lanes 2 and 3) compared with an oligoadenylated (A8, lanes 5 and 6) RNA14 substrate was analysed. Products were separated and visualized as described earlier. ( C ) The effect of LRPPRC/SLIRP complex (0.48 µ m ; lanes 3 and 5) on polyadenylation by p.N478D mutant (0.55 µ m ; lanes 4–5) poly(A) polymerase was compared with wild-type (0.55 µM; lanes 2–3) enzyme. The short unadenylated RNA14 A0 substrate (0.25 µ m ) was used. Products were separated and visualized as described earlier.

    Techniques Used: Activity Assay, Mutagenesis, Incubation

    The MTPAP 1432A > G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. ( A ) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C , unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A 0 ) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A 50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. ( B ) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. ( C ) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. ( D ) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t -test (ns * P
    Figure Legend Snippet: The MTPAP 1432A > G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. ( A ) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C , unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A 0 ) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A 50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. ( B ) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. ( C ) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. ( D ) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t -test (ns * P

    Techniques Used: Mutagenesis, Isolation, Transduction, Migration, SDS Page, Imaging, Blue Native PAGE

    In vitro polyadenylation activity of mtPAP. ( A ) Polyadenylation activity of recombinant wild-type (WT; lanes 2–4) and mutant (p.N478D; lanes 6–8) mitochondrial poly(A) polymerase (0.55 µ m ) was determined with increasing ATP concentrations. The RNA substrate was an unadenylated 277-nt 3′ fragment of MTND3 (0.25 µ m ). The right hand panel contains an IVT RNA artefact (500 nt) present in the absence of mtPAP (lane 5). Reactions were quenched with 90% formamide/1× TBE, separated through a 6% polyacrylamide/8.3 m urea gel, then stained with SYBR gold and visualized by scanning with a Typhoon FLA 9500 instrument. ( B ) Short RNAs (0.25 µ m ) corresponding to the final 40 nucleotides of RNA14 with (A8, lanes 5–8) or without (A0, lanes 1–4) an oligo(A8) addition were used as templates for polyadenylation by recombinant wild-type (WT; lanes 2 and 6) or mutant (p.N478D; lanes 3 and 7) mtPAP (0.55 µ m ). An equal amount of BSA was added in a parallel experiment as a control (lanes 4 and 8). Products were separated through 15% polyacrylamide/8.3 m urea gel and visualized as in (A). ( C ) Increasing amounts of wild-type mtPAP (34 n m to 0.55 µ m ) were added to the short RNA14 A0 (0.25 µ m ) template in the presence (lanes 8–12) or absence (lanes 2–6) of LRPRRC/SLIRP complex. A higher molecular species (*) of varying intensity was observed with wild-type mtPAP. Products were separated and visualized as in (B).
    Figure Legend Snippet: In vitro polyadenylation activity of mtPAP. ( A ) Polyadenylation activity of recombinant wild-type (WT; lanes 2–4) and mutant (p.N478D; lanes 6–8) mitochondrial poly(A) polymerase (0.55 µ m ) was determined with increasing ATP concentrations. The RNA substrate was an unadenylated 277-nt 3′ fragment of MTND3 (0.25 µ m ). The right hand panel contains an IVT RNA artefact (500 nt) present in the absence of mtPAP (lane 5). Reactions were quenched with 90% formamide/1× TBE, separated through a 6% polyacrylamide/8.3 m urea gel, then stained with SYBR gold and visualized by scanning with a Typhoon FLA 9500 instrument. ( B ) Short RNAs (0.25 µ m ) corresponding to the final 40 nucleotides of RNA14 with (A8, lanes 5–8) or without (A0, lanes 1–4) an oligo(A8) addition were used as templates for polyadenylation by recombinant wild-type (WT; lanes 2 and 6) or mutant (p.N478D; lanes 3 and 7) mtPAP (0.55 µ m ). An equal amount of BSA was added in a parallel experiment as a control (lanes 4 and 8). Products were separated through 15% polyacrylamide/8.3 m urea gel and visualized as in (A). ( C ) Increasing amounts of wild-type mtPAP (34 n m to 0.55 µ m ) were added to the short RNA14 A0 (0.25 µ m ) template in the presence (lanes 8–12) or absence (lanes 2–6) of LRPRRC/SLIRP complex. A higher molecular species (*) of varying intensity was observed with wild-type mtPAP. Products were separated and visualized as in (B).

    Techniques Used: In Vitro, Activity Assay, Recombinant, Mutagenesis, Staining

    13) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿ †"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01804-07

    Herpesvirus miRNAs share seed sequence homology with human miRNAs. (A) miRNA sequences from KSHV, EBV, and human cytomegalovirus were aligned with sequences in the human miRNA database. Shown are those viral miRNAs exhibiting the greatest sequence homology to human miRNAs. (B) miR-155 is expressed from exon 3 of the non-protein-coding BIC. Primers (forward [fwd] and reverse [rev]) for RT-PCR analysis of BIC expression are indicated. (C) RT-PCR analysis of BIC expression in KSHV-infected PEL cell lines. The RAJI cell line, an EBV-infected BL cell line, was used as a positive control. H2O indicates the no-template control. (D) Northern blot analysis of KSHV-miR-K12-11 and hsa-miR-155 expression in PEL and BL cells. Twenty-five micrograms of total RNA was loaded per lane and hybridized to probes for either miR-K12-11 or miR-155.
    Figure Legend Snippet: Herpesvirus miRNAs share seed sequence homology with human miRNAs. (A) miRNA sequences from KSHV, EBV, and human cytomegalovirus were aligned with sequences in the human miRNA database. Shown are those viral miRNAs exhibiting the greatest sequence homology to human miRNAs. (B) miR-155 is expressed from exon 3 of the non-protein-coding BIC. Primers (forward [fwd] and reverse [rev]) for RT-PCR analysis of BIC expression are indicated. (C) RT-PCR analysis of BIC expression in KSHV-infected PEL cell lines. The RAJI cell line, an EBV-infected BL cell line, was used as a positive control. H2O indicates the no-template control. (D) Northern blot analysis of KSHV-miR-K12-11 and hsa-miR-155 expression in PEL and BL cells. Twenty-five micrograms of total RNA was loaded per lane and hybridized to probes for either miR-K12-11 or miR-155.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Positive Control, Northern Blot

    14) Product Images from "An AlgU-Regulated Antisense Transcript Encoded within the Pseudomonas syringaefleQ Gene Has a Positive Effect on Motility"

    Article Title: An AlgU-Regulated Antisense Transcript Encoded within the Pseudomonas syringaefleQ Gene Has a Positive Effect on Motility

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00576-17

    Analysis of AlgU-dependent antisense transcript expressed within the fleQ gene. (A) RNA-seq and ChIP-seq results at the fleQ locus. Strand-specific RNA-seq analysis of transcriptomes produced by P. syringae pv. tomato DC3000 Δ algU mucAB cells containing an AlgU expression construct (+AlgU) or the empty-vector control (-Cntl) show the location of antisense transcription in the fleQ gene (dashed box). ChIP-seq analysis of P. syringae pv. tomato DC3000 Δ algU mucAB cells containing an AlgU-Flag expression construct (+AlgU-Flag) or the empty-vector control (-Cntl) shows AlgU-Flag-dependent enrichment of sequences directly upstream of the location of fleQ as . The number of reads on the y axis indicates the number of sequence reads mapped per nucleotide position. The red and blue profiles indicate sequences matching the sense and antisense strands, respectively. (B) Strand-specific RT-PCR was used to test for the fleQ as transcript in cells containing either the AlgU expression plasmid or the empty-vector control. PCR amplification required the product of strand-specific reverse transcription of the fleQ as or the fleQ transcript. Primers complementary to either the fleQ as or the fleQ transcript were used to reverse transcribe a segment of each transcript that served as the template in a PCR to detect the cDNA products. The numbered arrows refer to oligonucleotide primers (see Table S3 in the supplemental material). (C) Agarose gel showing reverse transcriptase (RT)-dependent PCR products of fleQ as , which were obtained only from cells expressing AlgU. In contrast, the fleQ sense transcripts were detected in cells containing AlgU expression and the empty vector (EV). P. syringae pv. tomato DC3000 genomic DNA (gDNA) was a positive control for the gene-specific PCR.
    Figure Legend Snippet: Analysis of AlgU-dependent antisense transcript expressed within the fleQ gene. (A) RNA-seq and ChIP-seq results at the fleQ locus. Strand-specific RNA-seq analysis of transcriptomes produced by P. syringae pv. tomato DC3000 Δ algU mucAB cells containing an AlgU expression construct (+AlgU) or the empty-vector control (-Cntl) show the location of antisense transcription in the fleQ gene (dashed box). ChIP-seq analysis of P. syringae pv. tomato DC3000 Δ algU mucAB cells containing an AlgU-Flag expression construct (+AlgU-Flag) or the empty-vector control (-Cntl) shows AlgU-Flag-dependent enrichment of sequences directly upstream of the location of fleQ as . The number of reads on the y axis indicates the number of sequence reads mapped per nucleotide position. The red and blue profiles indicate sequences matching the sense and antisense strands, respectively. (B) Strand-specific RT-PCR was used to test for the fleQ as transcript in cells containing either the AlgU expression plasmid or the empty-vector control. PCR amplification required the product of strand-specific reverse transcription of the fleQ as or the fleQ transcript. Primers complementary to either the fleQ as or the fleQ transcript were used to reverse transcribe a segment of each transcript that served as the template in a PCR to detect the cDNA products. The numbered arrows refer to oligonucleotide primers (see Table S3 in the supplemental material). (C) Agarose gel showing reverse transcriptase (RT)-dependent PCR products of fleQ as , which were obtained only from cells expressing AlgU. In contrast, the fleQ sense transcripts were detected in cells containing AlgU expression and the empty vector (EV). P. syringae pv. tomato DC3000 genomic DNA (gDNA) was a positive control for the gene-specific PCR.

    Techniques Used: RNA Sequencing Assay, Chromatin Immunoprecipitation, Produced, Expressing, Construct, Plasmid Preparation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Positive Control

    15) Product Images from "Site-specific DICER and DROSHA RNA products control the DNA damage response"

    Article Title: Site-specific DICER and DROSHA RNA products control the DNA damage response

    Journal: Nature

    doi: 10.1038/nature11179

    Irradiation-induced DDR foci are sensitive to RNase A treatment and are restored by small and DICER dependent RNAs a. Irradiated HeLa cells (2 Gy) were treated with PBS (-) or RNase A (+) and probed for 53BP1, pATM, pS/TQ, MDC1 and γH2AX foci. Histogram shows the percentage of cells positive for DDR foci. b. 100, 50 or 20 ng of gel-extracted total RNA and 50 ng of RNA extracted from each gel fraction ( > 100, 35-100 and 20-35 nt) were used for DDR foci reconstitution after RNase treatment. c. 53BP1, pS/TQ and pATM foci are restored in RNase-treated cells when incubated with RNA of wild-type cells but not with RNA of DICER exon5 cells or tRNA. e. Histogram shows the percentage of cells positive for DDR foci. Error bars indicate s.e.m. (n ≥3). Differences are statistically significant (* p-value
    Figure Legend Snippet: Irradiation-induced DDR foci are sensitive to RNase A treatment and are restored by small and DICER dependent RNAs a. Irradiated HeLa cells (2 Gy) were treated with PBS (-) or RNase A (+) and probed for 53BP1, pATM, pS/TQ, MDC1 and γH2AX foci. Histogram shows the percentage of cells positive for DDR foci. b. 100, 50 or 20 ng of gel-extracted total RNA and 50 ng of RNA extracted from each gel fraction ( > 100, 35-100 and 20-35 nt) were used for DDR foci reconstitution after RNase treatment. c. 53BP1, pS/TQ and pATM foci are restored in RNase-treated cells when incubated with RNA of wild-type cells but not with RNA of DICER exon5 cells or tRNA. e. Histogram shows the percentage of cells positive for DDR foci. Error bars indicate s.e.m. (n ≥3). Differences are statistically significant (* p-value

    Techniques Used: Irradiation, Incubation

    Site-specific DDR focus formation is RNase A-sensitive and can be restored by site-specific RNA in a MRN-dependent manner a. Cut NIH2/4 cells display a 53BP1 and γH2AX focus colocalizing with Cherry-Lac focus. 53BP1, but not γH2AX, focus is sensitive to RNase A and is restored by incubation with total RNA. b. Histogram shows the percentage of cells in which 53BP1 and Cherry-Lac foci co-localize. Addition of 50, 200, or 800 ng of RNA purified from cut NIH2/4 rescues 53BP1 foci formation in a dose-dependent manner. c. RNA purified from cut NIH2/4 restores 53BP1 focus while RNA from parental cells expressing I- Sce I does not. d, e. RNase A-treated cut NIH2/4 cells were incubated with RNA from cut NIH2/4 cells, or parental ones, to test 53BP1 or pATM focus reformation in the presence of the MRN inhibitor mirin (100 μM). Histogram shows the percentage of cells positive for DDR focus. Error bars indicate s.e.m. (n ≥3). Differences are statistically significant (* p-value
    Figure Legend Snippet: Site-specific DDR focus formation is RNase A-sensitive and can be restored by site-specific RNA in a MRN-dependent manner a. Cut NIH2/4 cells display a 53BP1 and γH2AX focus colocalizing with Cherry-Lac focus. 53BP1, but not γH2AX, focus is sensitive to RNase A and is restored by incubation with total RNA. b. Histogram shows the percentage of cells in which 53BP1 and Cherry-Lac foci co-localize. Addition of 50, 200, or 800 ng of RNA purified from cut NIH2/4 rescues 53BP1 foci formation in a dose-dependent manner. c. RNA purified from cut NIH2/4 restores 53BP1 focus while RNA from parental cells expressing I- Sce I does not. d, e. RNase A-treated cut NIH2/4 cells were incubated with RNA from cut NIH2/4 cells, or parental ones, to test 53BP1 or pATM focus reformation in the presence of the MRN inhibitor mirin (100 μM). Histogram shows the percentage of cells positive for DDR focus. Error bars indicate s.e.m. (n ≥3). Differences are statistically significant (* p-value

    Techniques Used: Incubation, Purification, Expressing

    Chemically synthesized small RNAs and  in vitro  generated DICER RNA products are sufficient to restore DDR focus formation in RNase A-treated cells in a sequence-specific manner a.  Chemically synthesized oligonucleotides were annealed and were tested to restore DDR focus formation in RNase A-treated cut NIH2/4 cells. Mixed with a constant amount (800 ng) of parental cells RNA, a concentrations range (1 ng/μl – 1 fg/μl, ten-fold dilution steps) of locus-specific or GFP RNAs was used. Locus-specific synthetic RNAs (down to 100 fg/μl) allow site-specific DDR activation.  b . Short double-stranded RNAs generated by recombinant DICER were tested to restore DDR focus formation in RNase A-treated cut NIH2/4 cells. 1 ng/μl RNAs were tested mixed with 800 ng of parental cells RNA. Locus-specific DICER RNAs, but not control RNAs, allow site-specific DDR activation. Histograms show the percentage of cells positive for DDR focus. Error bars indicate s.e.m. (n ≥3). Differences are statistically significant (* p-value
    Figure Legend Snippet: Chemically synthesized small RNAs and in vitro generated DICER RNA products are sufficient to restore DDR focus formation in RNase A-treated cells in a sequence-specific manner a. Chemically synthesized oligonucleotides were annealed and were tested to restore DDR focus formation in RNase A-treated cut NIH2/4 cells. Mixed with a constant amount (800 ng) of parental cells RNA, a concentrations range (1 ng/μl – 1 fg/μl, ten-fold dilution steps) of locus-specific or GFP RNAs was used. Locus-specific synthetic RNAs (down to 100 fg/μl) allow site-specific DDR activation. b . Short double-stranded RNAs generated by recombinant DICER were tested to restore DDR focus formation in RNase A-treated cut NIH2/4 cells. 1 ng/μl RNAs were tested mixed with 800 ng of parental cells RNA. Locus-specific DICER RNAs, but not control RNAs, allow site-specific DDR activation. Histograms show the percentage of cells positive for DDR focus. Error bars indicate s.e.m. (n ≥3). Differences are statistically significant (* p-value

    Techniques Used: Synthesized, In Vitro, Generated, Sequencing, Activation Assay, Recombinant

    16) Product Images from "Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)"

    Article Title: Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt634

    Expression, localization and tRNA Asp(GUC) methylation activity of DnmA under various conditions. ( A ) dnmA expression is regulated during the cell cycle. Cells were arrested in the cell cycle by cold treatment and then released by transfer to 22°C. Cells were counted every 30 min (grey dots and grey line), and samples were taken for qPCR (black bars). After cell division, dnmA expression increased ∼5-fold and then rapidly declined to basal levels. Normalization was done on vegetative growing cells. dnmA expression is only shown from 3 to 4.5 h during recovery. ( B ) DnmA is lost from the nucleus during mitosis (arrow). The three cells on the right are in S-phase as indicated by the RFP-PCNA marker and accumulate DnmA in the nucleus (for further details see movie in Supplementary Material ). ( C ) Relative quantification of dnmA expression levels in AX2 cells after 2.5 h recovery from cold shock at 4°C. Expression of dnmA increased > 40-fold and returned within 30 min to basal levels (n = 3). ( D ) At 16 h of development, dnmA expression increased ∼46-fold in the NC4 strain. In AX2 cells, expression increased only ∼5-fold. ( E ) At 16 h of development, in vivo methylation of C38 in tRNA Asp(GUC) increased up to 75% in the D. discoideum strain NC4, whereas no significant increase was observed in AX2 cells.
    Figure Legend Snippet: Expression, localization and tRNA Asp(GUC) methylation activity of DnmA under various conditions. ( A ) dnmA expression is regulated during the cell cycle. Cells were arrested in the cell cycle by cold treatment and then released by transfer to 22°C. Cells were counted every 30 min (grey dots and grey line), and samples were taken for qPCR (black bars). After cell division, dnmA expression increased ∼5-fold and then rapidly declined to basal levels. Normalization was done on vegetative growing cells. dnmA expression is only shown from 3 to 4.5 h during recovery. ( B ) DnmA is lost from the nucleus during mitosis (arrow). The three cells on the right are in S-phase as indicated by the RFP-PCNA marker and accumulate DnmA in the nucleus (for further details see movie in Supplementary Material ). ( C ) Relative quantification of dnmA expression levels in AX2 cells after 2.5 h recovery from cold shock at 4°C. Expression of dnmA increased > 40-fold and returned within 30 min to basal levels (n = 3). ( D ) At 16 h of development, dnmA expression increased ∼46-fold in the NC4 strain. In AX2 cells, expression increased only ∼5-fold. ( E ) At 16 h of development, in vivo methylation of C38 in tRNA Asp(GUC) increased up to 75% in the D. discoideum strain NC4, whereas no significant increase was observed in AX2 cells.

    Techniques Used: Expressing, Methylation, Activity Assay, Real-time Polymerase Chain Reaction, Marker, In Vivo

    In vitro methylation of tRNA Gly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3 H-labelled methylated tRNAs. As a control tRNA Asp(GUC) methylation is shown.
    Figure Legend Snippet: In vitro methylation of tRNA Gly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3 H-labelled methylated tRNAs. As a control tRNA Asp(GUC) methylation is shown.

    Techniques Used: In Vitro, Methylation, Recombinant, Staining

    Ex vivo methylation and blocking assay. ( A ) Methylation of in vitro transcribed tRNA Asp(GUC) was completely blocked when a complementary antisense oligo was hybridized. ( B ) Using enriched small RNA from dnmA KO cells, ex vivo methylation in the tRNA size class was differentially lost when antisense oligos to tRNA Asp(GUC) and tRNA Glu(UUC) were hybridized before the methylation reaction. The oligo against tRNA Glu(UUC) also covers tRNA Glu(CUC) with minor mismatches (see Supplementary Figure S2 ). Even with both oligos, a significant amount of 3 H incorporation still remained. The upper panel shows the ethidiumbromide stained gel to demonstrate equal loading, the lower panel shows the fluorogram. The arrow marks a band that was specifically lost when the anti tRNA Glu(UUC) oligo was used.
    Figure Legend Snippet: Ex vivo methylation and blocking assay. ( A ) Methylation of in vitro transcribed tRNA Asp(GUC) was completely blocked when a complementary antisense oligo was hybridized. ( B ) Using enriched small RNA from dnmA KO cells, ex vivo methylation in the tRNA size class was differentially lost when antisense oligos to tRNA Asp(GUC) and tRNA Glu(UUC) were hybridized before the methylation reaction. The oligo against tRNA Glu(UUC) also covers tRNA Glu(CUC) with minor mismatches (see Supplementary Figure S2 ). Even with both oligos, a significant amount of 3 H incorporation still remained. The upper panel shows the ethidiumbromide stained gel to demonstrate equal loading, the lower panel shows the fluorogram. The arrow marks a band that was specifically lost when the anti tRNA Glu(UUC) oligo was used.

    Techniques Used: Ex Vivo, Methylation, Blocking Assay, In Vitro, Staining

    Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. ( A ) Examples of time courses on covalent complex formation with tRNA Asp(GUC) and tRNA Glu(UUC) . ( B ) Complex bands in A were quantified and presented on a time scale.
    Figure Legend Snippet: Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. ( A ) Examples of time courses on covalent complex formation with tRNA Asp(GUC) and tRNA Glu(UUC) . ( B ) Complex bands in A were quantified and presented on a time scale.

    Techniques Used:

    tRNA and tRNA fragments associated with DnmA-GFP. After UV crosslinking, RNA bound to DnmA was co-immunoprecipitated under denaturing conditions (CLIP). The number of normalised reads for tRNAs detected by Illumina sequencing is shown for DnmA CLIP and for the control CLIP with GFP. In addition to full-length tRNAs, four classes of tRNA fragments were found, and these are indicated by different shading of the bars. Size and localization of fragments is shown schematically on the simplified clover leaf structure. tRNAs that are not significantly enriched with DnmA are shown in Supplementary Figure S6 . In case of multiple gene copys resulting in the same RNA transcript, the sequencing reads were mapped to the first copy in the genome starting from chromsome 1. Only this copy is listed. Sequences and detailed information on gene copies and potential isoacceptors are listed in Supplementary Table S2 .
    Figure Legend Snippet: tRNA and tRNA fragments associated with DnmA-GFP. After UV crosslinking, RNA bound to DnmA was co-immunoprecipitated under denaturing conditions (CLIP). The number of normalised reads for tRNAs detected by Illumina sequencing is shown for DnmA CLIP and for the control CLIP with GFP. In addition to full-length tRNAs, four classes of tRNA fragments were found, and these are indicated by different shading of the bars. Size and localization of fragments is shown schematically on the simplified clover leaf structure. tRNAs that are not significantly enriched with DnmA are shown in Supplementary Figure S6 . In case of multiple gene copys resulting in the same RNA transcript, the sequencing reads were mapped to the first copy in the genome starting from chromsome 1. Only this copy is listed. Sequences and detailed information on gene copies and potential isoacceptors are listed in Supplementary Table S2 .

    Techniques Used: Immunoprecipitation, Cross-linking Immunoprecipitation, Sequencing

    In vitro methylation of tRNA Asp(GUC) . ( A ) In vitro methylation of tRNA Asp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl 2 and by hDnmt2 at 5 mM Mg 2+ . The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3 H-Me in the tRNAs. Reactions were run for the times indicated. ( B ) In vivo methylation of cytosines in tRNA Asp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNA Asp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. ( C ) In vitro methylation of small enriched RNA of a dnmA KO strain ( ex vivo methylation). The methylation reaction was done for the times indicated.
    Figure Legend Snippet: In vitro methylation of tRNA Asp(GUC) . ( A ) In vitro methylation of tRNA Asp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl 2 and by hDnmt2 at 5 mM Mg 2+ . The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3 H-Me in the tRNAs. Reactions were run for the times indicated. ( B ) In vivo methylation of cytosines in tRNA Asp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNA Asp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. ( C ) In vitro methylation of small enriched RNA of a dnmA KO strain ( ex vivo methylation). The methylation reaction was done for the times indicated.

    Techniques Used: In Vitro, Methylation, Staining, In Vivo, Methylation Sequencing, Sequencing, Ex Vivo

    17) Product Images from "De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function"

    Article Title: De Novo Prediction of PTBP1 Binding and Splicing Targets Reveals Unexpected Features of Its RNA Recognition and Function

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1003442

    Scheme of the PTBP1 splicing regulation model and its application to an exon in Ptbp3 . A . The PTBP1 splicing regulation model was trained on known PTBP1-regulated and non-regulated exons and used to predict new PTBP1-dependent exons. Prediction results were compared to changes in exon inclusion (PSI) measured by RT-PCR and RNA-seq. An exon from Ptbp3 is presented as a prediction example. From intron and exon sequences, PTBP1 binding scores and 3′ splice site strength were calculated and fed into the regulation model. B . The model predicts exon 2 of Ptbp3 as repressed by PTBP1 with high probability (0.89). Ptbp1 knockdown in mouse neuroblastoma cells ( N2A ) confirmed de-repression of the exon (from PSI = 45 to PSI = 70).
    Figure Legend Snippet: Scheme of the PTBP1 splicing regulation model and its application to an exon in Ptbp3 . A . The PTBP1 splicing regulation model was trained on known PTBP1-regulated and non-regulated exons and used to predict new PTBP1-dependent exons. Prediction results were compared to changes in exon inclusion (PSI) measured by RT-PCR and RNA-seq. An exon from Ptbp3 is presented as a prediction example. From intron and exon sequences, PTBP1 binding scores and 3′ splice site strength were calculated and fed into the regulation model. B . The model predicts exon 2 of Ptbp3 as repressed by PTBP1 with high probability (0.89). Ptbp1 knockdown in mouse neuroblastoma cells ( N2A ) confirmed de-repression of the exon (from PSI = 45 to PSI = 70).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, RNA Sequencing Assay, Binding Assay

    Validation of novel PTBP1-repressed exons by RT-PCR. A . Candidate PTBP1-repressed exons with probability greater than 0.65 were validated by RT-PCR following Ptbp1 knockdown. Data shown are averages ± standard error of PSI (Percent of Spliced In) from biological triplicates. Statistical analysis was performed using paired one-tailed Student's t-test (p-values
    Figure Legend Snippet: Validation of novel PTBP1-repressed exons by RT-PCR. A . Candidate PTBP1-repressed exons with probability greater than 0.65 were validated by RT-PCR following Ptbp1 knockdown. Data shown are averages ± standard error of PSI (Percent of Spliced In) from biological triplicates. Statistical analysis was performed using paired one-tailed Student's t-test (p-values

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, One-tailed Test

    Validation of the PTBP1 binding model. A . To validate binding scores, thirteen RNAs with various PTBP1 binding scores were transcribed in vitro and subjected to binding assay. Apparent Kd 's (dissociation constant) were highly negatively correlated with PTBP1 binding scores (Pearson correlation = −0.9). B . Four RNA sequences with predicted PTBP1 binding scores (Full data binding data in Figure S3 ). Potential PTBP1 binding sites are underlined and in bold. Experimental binding affinities were assessed by electrophoretic mobility shift of RNA by PTBP1 and compared with prediction scores. Apparent dissociation constants ( Kd ) were defined as the concentration at which half the protein was bound to RNA.
    Figure Legend Snippet: Validation of the PTBP1 binding model. A . To validate binding scores, thirteen RNAs with various PTBP1 binding scores were transcribed in vitro and subjected to binding assay. Apparent Kd 's (dissociation constant) were highly negatively correlated with PTBP1 binding scores (Pearson correlation = −0.9). B . Four RNA sequences with predicted PTBP1 binding scores (Full data binding data in Figure S3 ). Potential PTBP1 binding sites are underlined and in bold. Experimental binding affinities were assessed by electrophoretic mobility shift of RNA by PTBP1 and compared with prediction scores. Apparent dissociation constants ( Kd ) were defined as the concentration at which half the protein was bound to RNA.

    Techniques Used: Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Concentration Assay

    Sequence characteristics of PTBP1-dependent alternatively spliced exons. A . An RNA map shows enrichment of predicted PTBP1 binding sites near PTBP1-dependent exons. The Y-axis plots average density of predicted PTBP1 binding states within a 24 nt window; the length of overlap between two adjacent windows was 8 nt. B . To assess PTBP1 binding signatures of individual exons, known PTBP1 regulated exons were clustered by their PTBP1 binding score profiles and visualized as heat maps. These heat maps indicate wide variation in the positions of PTBP1 binding sites between individual exons. C . Four sequence features including the PTBP1 binding scores and 3′ splice site strength show statistically significant differences between regulated and control exon groups (one-tailed Student's t-tests).
    Figure Legend Snippet: Sequence characteristics of PTBP1-dependent alternatively spliced exons. A . An RNA map shows enrichment of predicted PTBP1 binding sites near PTBP1-dependent exons. The Y-axis plots average density of predicted PTBP1 binding states within a 24 nt window; the length of overlap between two adjacent windows was 8 nt. B . To assess PTBP1 binding signatures of individual exons, known PTBP1 regulated exons were clustered by their PTBP1 binding score profiles and visualized as heat maps. These heat maps indicate wide variation in the positions of PTBP1 binding sites between individual exons. C . Four sequence features including the PTBP1 binding scores and 3′ splice site strength show statistically significant differences between regulated and control exon groups (one-tailed Student's t-tests).

    Techniques Used: Sequencing, Binding Assay, One-tailed Test

    PTBP1 binding model. A . Scheme of the PTBP1 binding model. The two-state HMM model was trained on PTBP1 bound RNA sequences (48,604 clusters) from published PTBP1-CLIP experiments. Triplets from these CLIP clusters were predictive of two states, with all of the pyrimidine triplets preferred by State 1. The diagram presents the structure of the PTBP1 HMM (Hidden Markov Model) and its trained transition probabilities. B . The probabilities that triplets are seen states 1 or 2 (emission probabilities) are plotted in black and gray bars, respectively. Asterisks indicate G containing pyrimidine triplets.
    Figure Legend Snippet: PTBP1 binding model. A . Scheme of the PTBP1 binding model. The two-state HMM model was trained on PTBP1 bound RNA sequences (48,604 clusters) from published PTBP1-CLIP experiments. Triplets from these CLIP clusters were predictive of two states, with all of the pyrimidine triplets preferred by State 1. The diagram presents the structure of the PTBP1 HMM (Hidden Markov Model) and its trained transition probabilities. B . The probabilities that triplets are seen states 1 or 2 (emission probabilities) are plotted in black and gray bars, respectively. Asterisks indicate G containing pyrimidine triplets.

    Techniques Used: Binding Assay, Cross-linking Immunoprecipitation

    Large-scale validation of novel PTBP1-repressed exons by RNA-seq. A . Validation of the PTBP1 splicing model using RNA-seq. After Ptbp1 knockdown, we performed RNA-seq experiments and estimated changes in PSI (Percent of Spliced In) for 573 cassette exons. The graph shows average delta PSI values for exons, grouped by their probabilities to be repressed by PTBP1. The number of exons in the corresponding probability bin is given by n. P-values were calculated from one-tailed Student's t-test. B . A genome browser screenshot of a novel PTBP1-regulated exon: exon 2 of the Kcnq2 gene. For whole internal mouse exons, we created custom genome browser tracks to visualize the PTBP1 splicing model and mapped RNA seq reads.
    Figure Legend Snippet: Large-scale validation of novel PTBP1-repressed exons by RNA-seq. A . Validation of the PTBP1 splicing model using RNA-seq. After Ptbp1 knockdown, we performed RNA-seq experiments and estimated changes in PSI (Percent of Spliced In) for 573 cassette exons. The graph shows average delta PSI values for exons, grouped by their probabilities to be repressed by PTBP1. The number of exons in the corresponding probability bin is given by n. P-values were calculated from one-tailed Student's t-test. B . A genome browser screenshot of a novel PTBP1-regulated exon: exon 2 of the Kcnq2 gene. For whole internal mouse exons, we created custom genome browser tracks to visualize the PTBP1 splicing model and mapped RNA seq reads.

    Techniques Used: RNA Sequencing Assay, One-tailed Test

    18) Product Images from "SUMOylation modulates the LIN28A‐let‐7 signaling pathway in response to cellular stresses in cancer cells"

    Article Title: SUMOylation modulates the LIN28A‐let‐7 signaling pathway in response to cellular stresses in cancer cells

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12694

    SUMOylation of LIN28A exacerbates its inhibition of let‐7 biogenesis. (A) Disrupting SUMOylation system interferences let‐7 biogenesis. HeLa‐shpLKO.1, HeLa‐shSENP1, and HeLa‐shUBC9 cells were transiently transfected with HA‐LIN28A. 48 h after transfection, the expression levels of endogenous let‐7a and let‐7c were detected by northern blotting with indicated probes. The knockdown efficiency of SENP1 and UBC9 was detected by western blot with indicated antibodies. The SENP1 and UBC9 bands were quantified by imagej software and normalized with Tubulin. (B) Stable knockout of SENP1 leads to down‐regulation of mature let‐7a levels. 293T cells and 293T SENP1 −/− cells were transiently transfected with HA‐LIN28A. The expression level of endogenous let‐7a was analyzed by northern blotting. The effects of knockout of SENP1 were detected by western blotting with indicated antibodies. (C) SUMO1 modification of LIN28A enhances its inhibition of let‐7s biogenesis. 293T cells were co‐transfected with HA‐LIN28A and human pri‐let‐7c, pre‐let‐7a‐1, or pre‐let‐7g, with or without His‐SUMO1, as indicated. 48 h after transfection, RNAs were extracted and separated on 20% polyacrylamide 8 m urea gels. U6 RNA was used as a control. (D) Truncated forms lacking K15 do not inhibit let‐7a biogenesis. 293T cells were transiently transfected with HA‐LIN28A and truncated forms as indicated. Northern blot was used to measure the expression levels of endogenous let‐7a. (E, F) Mutation K15R of LIN28A blocks its inhibition of let‐7 biogenesis. (E) 293T cells were transiently transfected with HA‐LIN28A or HA‐LIN28A‐K15R, along with pri‐let‐7a‐1 or pri‐let‐7c. (F) Stable DU145, MDA‐MB‐231, and T47D‐shLIN28A expressing the control vector, HA‐LIN28A, or HA‐LIN28A‐K15R were used for northern blotting analyses of let‐7s. The expression levels of LIN28A and LIN28A‐K15R were detected by western blotting with anti‐HA and anti‐LIN28A antibodies. All of let‐7 bands were quantified by imagej software and normalized with U6.
    Figure Legend Snippet: SUMOylation of LIN28A exacerbates its inhibition of let‐7 biogenesis. (A) Disrupting SUMOylation system interferences let‐7 biogenesis. HeLa‐shpLKO.1, HeLa‐shSENP1, and HeLa‐shUBC9 cells were transiently transfected with HA‐LIN28A. 48 h after transfection, the expression levels of endogenous let‐7a and let‐7c were detected by northern blotting with indicated probes. The knockdown efficiency of SENP1 and UBC9 was detected by western blot with indicated antibodies. The SENP1 and UBC9 bands were quantified by imagej software and normalized with Tubulin. (B) Stable knockout of SENP1 leads to down‐regulation of mature let‐7a levels. 293T cells and 293T SENP1 −/− cells were transiently transfected with HA‐LIN28A. The expression level of endogenous let‐7a was analyzed by northern blotting. The effects of knockout of SENP1 were detected by western blotting with indicated antibodies. (C) SUMO1 modification of LIN28A enhances its inhibition of let‐7s biogenesis. 293T cells were co‐transfected with HA‐LIN28A and human pri‐let‐7c, pre‐let‐7a‐1, or pre‐let‐7g, with or without His‐SUMO1, as indicated. 48 h after transfection, RNAs were extracted and separated on 20% polyacrylamide 8 m urea gels. U6 RNA was used as a control. (D) Truncated forms lacking K15 do not inhibit let‐7a biogenesis. 293T cells were transiently transfected with HA‐LIN28A and truncated forms as indicated. Northern blot was used to measure the expression levels of endogenous let‐7a. (E, F) Mutation K15R of LIN28A blocks its inhibition of let‐7 biogenesis. (E) 293T cells were transiently transfected with HA‐LIN28A or HA‐LIN28A‐K15R, along with pri‐let‐7a‐1 or pri‐let‐7c. (F) Stable DU145, MDA‐MB‐231, and T47D‐shLIN28A expressing the control vector, HA‐LIN28A, or HA‐LIN28A‐K15R were used for northern blotting analyses of let‐7s. The expression levels of LIN28A and LIN28A‐K15R were detected by western blotting with anti‐HA and anti‐LIN28A antibodies. All of let‐7 bands were quantified by imagej software and normalized with U6.

    Techniques Used: Inhibition, Transfection, Expressing, Northern Blot, Western Blot, Software, Knock-Out, Modification, Mutagenesis, Multiple Displacement Amplification, Plasmid Preparation

    SUMOylation of LIN28A augments its affinity with pre‐let‐7 in vitro . (A) Top: Schematic maps of r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 protein domains. Bottom: Coomassie Blue staining of purified r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 proteins. (B, C) Binding of r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 to synthetic preE‐let‐7a‐1 was assessed by EMSA with 5 n m of 5′‐end biotin‐labeled preE‐let‐7a‐1 (B) or preE‐let‐7g (C) and the indicated concentration of recombinant proteins. The band intensities were quantified by imagej software and presented as the fraction of bound preE‐let‐7g RNA in the plots. (D) SPR analysis of the direct binding of r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 to synthetic preE‐let‐7g using an Biacore T200 instrument. The binding affinity was determined by global fitting to a Langmuir 1 : 1 binding model within the biacore evaluation software. (E) The crystal structures of SUMO1 (PDB: 4WJQ ) and LIN28A‐preE‐Let‐7g (PDB: 3TS2 ) were blindly docked at ClusPro 2.0 docking server with a distance restrain of 20 Angstroms between Ca atoms of Gly96 of Sumo1 and Gln36 of LIN28A. The top solution from the server was selected for presentations here (left). The N‐terminal segment of LIN28A (residues 12–35), which was missing in the crystal structure, is modeled here as a helical structure with the sidechain of K15 (shown in sticks) covalently linked to the carboxyl group of Gly96 of SUMO1. The covalently linked SUMO1 (purple) on LIN28A (cyan) could readily interact with the backbone of preE‐let‐7g (brown) in the complex where the positively charged surface of SUMO1 (right) can form strong electrostatic interactions with the negatively charged phosphate groups of preE‐let‐7g. Therefore, the SUMOylated LIN28A would have higher binding affinity toward preE‐let‐7g. The electrostatic surface (right) of Sumo1 (with positively charged area shown in blue and negatively charged area shown in red) was calculated with software APBS, and the cartoon of the structures was generated using software pymol .
    Figure Legend Snippet: SUMOylation of LIN28A augments its affinity with pre‐let‐7 in vitro . (A) Top: Schematic maps of r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 protein domains. Bottom: Coomassie Blue staining of purified r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 proteins. (B, C) Binding of r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 to synthetic preE‐let‐7a‐1 was assessed by EMSA with 5 n m of 5′‐end biotin‐labeled preE‐let‐7a‐1 (B) or preE‐let‐7g (C) and the indicated concentration of recombinant proteins. The band intensities were quantified by imagej software and presented as the fraction of bound preE‐let‐7g RNA in the plots. (D) SPR analysis of the direct binding of r.LIN28A‐∆14 and r.SUMO1‐LIN28A‐∆14 to synthetic preE‐let‐7g using an Biacore T200 instrument. The binding affinity was determined by global fitting to a Langmuir 1 : 1 binding model within the biacore evaluation software. (E) The crystal structures of SUMO1 (PDB: 4WJQ ) and LIN28A‐preE‐Let‐7g (PDB: 3TS2 ) were blindly docked at ClusPro 2.0 docking server with a distance restrain of 20 Angstroms between Ca atoms of Gly96 of Sumo1 and Gln36 of LIN28A. The top solution from the server was selected for presentations here (left). The N‐terminal segment of LIN28A (residues 12–35), which was missing in the crystal structure, is modeled here as a helical structure with the sidechain of K15 (shown in sticks) covalently linked to the carboxyl group of Gly96 of SUMO1. The covalently linked SUMO1 (purple) on LIN28A (cyan) could readily interact with the backbone of preE‐let‐7g (brown) in the complex where the positively charged surface of SUMO1 (right) can form strong electrostatic interactions with the negatively charged phosphate groups of preE‐let‐7g. Therefore, the SUMOylated LIN28A would have higher binding affinity toward preE‐let‐7g. The electrostatic surface (right) of Sumo1 (with positively charged area shown in blue and negatively charged area shown in red) was calculated with software APBS, and the cartoon of the structures was generated using software pymol .

    Techniques Used: In Vitro, Staining, Purification, Binding Assay, Labeling, Concentration Assay, Recombinant, Software, SPR Assay, Generated

    SUMOylation of LIN28A increases its binding to pre‐let‐7. (A) Knockout of SENP1 increases the interaction of LIN28A with pre‐let‐7a‐1. 293T cells and 293T SENP1 −/− cells transfected with HA‐LIN28A and pre‐let‐7a‐1 were lysed for RIP assay. (B) SUMO1 modification enhances the association of LIN28A with pre‐let‐7a‐1. 293T cells transfected with indicated plasmids were used for RIP assays. (C) DeSUMOylation by SENP1 suppresses the interaction of LIN28A with pre‐let‐7g. 293T cells transfected with indicated plasmids were used for RIP assays. (D) Cisplatin suppresses the interaction of LIN28A with pre‐let‐7g. DU145 stably expressing HA‐LIN28A cells or T47D‐shLIN28A re‐expressing HA‐LIN28A stable cells were treated with Cisplatin (10 µ m ) for 12 h, and then, RIP assays were performed. (E, F) Pre‐let‐7s bound to LIN28A‐WT were much more than that to SUMO‐site mutant LIN28A‐K15R. 293T cells transfected with HA‐LIN28A or HA‐LIN28A‐K15R together with indicated pre‐let‐7 plasmids were used for RIP assays (E). RIP analysis of RNAs associated with HA‐LIN28A or HA‐LIN28A‐K15R from DU145 and T47D stable cell lines (F). RIP assays were carried out with anti‐HA (E) or anti‐LIN28A (F) antibody. (G) RNA pull‐down assay using biotinylated pre‐let‐7g and lysates from 293T cells transiently expressing HA‐LIN28A or HA‐LIN28A‐K15R. RNA‐bound fraction (beads) and unbound fraction (supernatant) were detected by western blotting with anti‐HA antibody. LIN28A bands were quantified by imagej software. The schematic diagram of RNA pull‐down assay is presented. RNAs extracted from IP complexes were analyzed by qRT–PCR, all RNA signals of RIP were normalized to those of input, then presented by relative binding fold. All data for qRT–PCR are presented as the mean ± SD with triplicates or quadruplicate sets, differences between individual groups as indicated were analyzed using the t ‐test (two‐tailed and unpaired), and P values of
    Figure Legend Snippet: SUMOylation of LIN28A increases its binding to pre‐let‐7. (A) Knockout of SENP1 increases the interaction of LIN28A with pre‐let‐7a‐1. 293T cells and 293T SENP1 −/− cells transfected with HA‐LIN28A and pre‐let‐7a‐1 were lysed for RIP assay. (B) SUMO1 modification enhances the association of LIN28A with pre‐let‐7a‐1. 293T cells transfected with indicated plasmids were used for RIP assays. (C) DeSUMOylation by SENP1 suppresses the interaction of LIN28A with pre‐let‐7g. 293T cells transfected with indicated plasmids were used for RIP assays. (D) Cisplatin suppresses the interaction of LIN28A with pre‐let‐7g. DU145 stably expressing HA‐LIN28A cells or T47D‐shLIN28A re‐expressing HA‐LIN28A stable cells were treated with Cisplatin (10 µ m ) for 12 h, and then, RIP assays were performed. (E, F) Pre‐let‐7s bound to LIN28A‐WT were much more than that to SUMO‐site mutant LIN28A‐K15R. 293T cells transfected with HA‐LIN28A or HA‐LIN28A‐K15R together with indicated pre‐let‐7 plasmids were used for RIP assays (E). RIP analysis of RNAs associated with HA‐LIN28A or HA‐LIN28A‐K15R from DU145 and T47D stable cell lines (F). RIP assays were carried out with anti‐HA (E) or anti‐LIN28A (F) antibody. (G) RNA pull‐down assay using biotinylated pre‐let‐7g and lysates from 293T cells transiently expressing HA‐LIN28A or HA‐LIN28A‐K15R. RNA‐bound fraction (beads) and unbound fraction (supernatant) were detected by western blotting with anti‐HA antibody. LIN28A bands were quantified by imagej software. The schematic diagram of RNA pull‐down assay is presented. RNAs extracted from IP complexes were analyzed by qRT–PCR, all RNA signals of RIP were normalized to those of input, then presented by relative binding fold. All data for qRT–PCR are presented as the mean ± SD with triplicates or quadruplicate sets, differences between individual groups as indicated were analyzed using the t ‐test (two‐tailed and unpaired), and P values of

    Techniques Used: Binding Assay, Knock-Out, Transfection, Modification, Stable Transfection, Expressing, Mutagenesis, Pull Down Assay, Western Blot, Software, Quantitative RT-PCR, Two Tailed Test

    SUMOylation of LIN28A promotes pre‐let‐7 uridylation and inhibits pre‐let‐7 processing. (A, D) Schematic representation is shown for protein purification strategy from 293T cells transfected as indicated expressing plasmids. Purified Flag‐tagged LIN28A and SUMO1‐LIN28A (transfected with SUMO1/UBC9) proteins were immunoblotted with the indicated antibodies (A, right panel). (B, E) SUMOylation of LIN28A promotes pre‐let‐7 oligouridylation. For in vitro uridylation assay, purified Flag‐tagged LIN28A‐WT, SUMO1‐LIN28A (A), or LIN28A‐K15R (D) was incubated with in vitro transcribed uniformly biotin‐labeled pre‐let‐7g along with or without Flag‐TUT4, which was immunoprecipitated from 293T cells. After 30 min of reaction, RNAs were exacted from the reaction mixture and separated on 20% polyacrylamide 8 m urea gels, and then detected by northern blot. (C, F) SUMOylation of LIN28A inhibits pre‐let‐7 processing by DICER. For in vitro processing assay, transcribed in vitro uniformly biotin‐labeled pre‐let‐7g was incubated with purified Flag‐tagged LIN28A‐WT, SUMO1‐LIN28A (A), or LIN28A‐K15R (D) along with Flag‐HA‐DICER immunoprecipitated from 293T cells. After 60 min of reaction, RNAs were exacted from the reaction mixture and separated on 20% polyacrylamide 8 m urea gels and then detected by northern blot.
    Figure Legend Snippet: SUMOylation of LIN28A promotes pre‐let‐7 uridylation and inhibits pre‐let‐7 processing. (A, D) Schematic representation is shown for protein purification strategy from 293T cells transfected as indicated expressing plasmids. Purified Flag‐tagged LIN28A and SUMO1‐LIN28A (transfected with SUMO1/UBC9) proteins were immunoblotted with the indicated antibodies (A, right panel). (B, E) SUMOylation of LIN28A promotes pre‐let‐7 oligouridylation. For in vitro uridylation assay, purified Flag‐tagged LIN28A‐WT, SUMO1‐LIN28A (A), or LIN28A‐K15R (D) was incubated with in vitro transcribed uniformly biotin‐labeled pre‐let‐7g along with or without Flag‐TUT4, which was immunoprecipitated from 293T cells. After 30 min of reaction, RNAs were exacted from the reaction mixture and separated on 20% polyacrylamide 8 m urea gels, and then detected by northern blot. (C, F) SUMOylation of LIN28A inhibits pre‐let‐7 processing by DICER. For in vitro processing assay, transcribed in vitro uniformly biotin‐labeled pre‐let‐7g was incubated with purified Flag‐tagged LIN28A‐WT, SUMO1‐LIN28A (A), or LIN28A‐K15R (D) along with Flag‐HA‐DICER immunoprecipitated from 293T cells. After 60 min of reaction, RNAs were exacted from the reaction mixture and separated on 20% polyacrylamide 8 m urea gels and then detected by northern blot.

    Techniques Used: Protein Purification, Transfection, Expressing, Purification, In Vitro, Incubation, Labeling, Immunoprecipitation, Northern Blot

    19) Product Images from "Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)"

    Article Title: Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt634

    In vitro methylation of tRNA Gly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3 H-labelled methylated tRNAs. As a control tRNA Asp(GUC) methylation is shown.
    Figure Legend Snippet: In vitro methylation of tRNA Gly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3 H-labelled methylated tRNAs. As a control tRNA Asp(GUC) methylation is shown.

    Techniques Used: In Vitro, Methylation, Recombinant, Staining

    Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. ( A ) Examples of time courses on covalent complex formation with tRNA Asp(GUC) and tRNA Glu(UUC) . ( B ) Complex bands in A were quantified and presented on a time scale.
    Figure Legend Snippet: Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. ( A ) Examples of time courses on covalent complex formation with tRNA Asp(GUC) and tRNA Glu(UUC) . ( B ) Complex bands in A were quantified and presented on a time scale.

    Techniques Used:

    In vitro methylation of tRNA Asp(GUC) . ( A ) In vitro methylation of tRNA Asp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl 2 and by hDnmt2 at 5 mM Mg 2+ . The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3 H-Me in the tRNAs. Reactions were run for the times indicated. ( B ) In vivo methylation of cytosines in tRNA Asp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNA Asp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. ( C ) In vitro methylation of small enriched RNA of a dnmA KO strain ( ex vivo methylation). The methylation reaction was done for the times indicated.
    Figure Legend Snippet: In vitro methylation of tRNA Asp(GUC) . ( A ) In vitro methylation of tRNA Asp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl 2 and by hDnmt2 at 5 mM Mg 2+ . The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3 H-Me in the tRNAs. Reactions were run for the times indicated. ( B ) In vivo methylation of cytosines in tRNA Asp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNA Asp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. ( C ) In vitro methylation of small enriched RNA of a dnmA KO strain ( ex vivo methylation). The methylation reaction was done for the times indicated.

    Techniques Used: In Vitro, Methylation, Staining, In Vivo, Methylation Sequencing, Sequencing, Ex Vivo

    20) Product Images from "Hairpin structure within the 3?UTR of DNA polymerase ? mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1"

    Article Title: Hairpin structure within the 3?UTR of DNA polymerase ? mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm502

    The evolutionarily conserved part of the 3′UTR of the DNA polymerase β transcript forms a stable hairpin structure. ( A ) Structural analysis of 5′-end labeled 3′UTR of the Pol β transcript (208 nt) with use of the following probes: Pb ions (0.25, 0.5 and 1 mM); S1 nuclease (0.5, 1 and 2 units/μl (1 mM ZnCl 2 was present in each reaction); RNase T1 (0.5, 1 and 1.5 units/μl); RNase T2 (0.1, 0.2 and 0.4 unit/μl); RNase Cl3 (0.2, 0.4 and 0.6 unit/μl) and RNase A (0.4, 0.8 and 1.6 unit/μl). Lane Ci, control incubation (no probe added); lane F, formamide, statistical ladder; lane T, guanine-specific ladder obtained with T1 ribonuclease digestion. The positions of selected G residues are shown along the T1 ladder. Fragments of autoradiograms corresponding to sequences forming the structure modules M1, M2 and M3 are indicated. ( B ) Proposed secondary structure model of the 3′UTR of the Pol β transcript. Cleavage sites are indicated for each probe used (see figure inset for probe designations and cleavage intensity classification). Three RNA structure modules (M1, M2 and M3), names of loop regions (a–k) and Poly-A signal (122–127 nt) are also marked.
    Figure Legend Snippet: The evolutionarily conserved part of the 3′UTR of the DNA polymerase β transcript forms a stable hairpin structure. ( A ) Structural analysis of 5′-end labeled 3′UTR of the Pol β transcript (208 nt) with use of the following probes: Pb ions (0.25, 0.5 and 1 mM); S1 nuclease (0.5, 1 and 2 units/μl (1 mM ZnCl 2 was present in each reaction); RNase T1 (0.5, 1 and 1.5 units/μl); RNase T2 (0.1, 0.2 and 0.4 unit/μl); RNase Cl3 (0.2, 0.4 and 0.6 unit/μl) and RNase A (0.4, 0.8 and 1.6 unit/μl). Lane Ci, control incubation (no probe added); lane F, formamide, statistical ladder; lane T, guanine-specific ladder obtained with T1 ribonuclease digestion. The positions of selected G residues are shown along the T1 ladder. Fragments of autoradiograms corresponding to sequences forming the structure modules M1, M2 and M3 are indicated. ( B ) Proposed secondary structure model of the 3′UTR of the Pol β transcript. Cleavage sites are indicated for each probe used (see figure inset for probe designations and cleavage intensity classification). Three RNA structure modules (M1, M2 and M3), names of loop regions (a–k) and Poly-A signal (122–127 nt) are also marked.

    Techniques Used: Labeling, Incubation

    21) Product Images from "Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9"

    Article Title: Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9

    Journal: eLife

    doi: 10.7554/eLife.18825

    In vitro interaction of Nup98 and DHX9. ( A ) Anti-DHX9 antibodies coupled to beads were used to immobilize recombinant DHX9. Bead-bound DHX9 was then incubated with recombinant Nup98 or GST in the presence or absence of RNA (poly I:C), RNase A, or buffer alone. Bound proteins were analyzed by Western blots using the indicated antibodies (below images). The top row of panels shows the DHX9 bait bound to beads. The bottom row of panels shows GST and Nup98 that bound to DHX9 under the indicated conditions. Asterisks denote positions of DHX9 and Nup98. The positions of molecular mass markers (shown in kDa) are indicated on the left and right. ( B ) Example images of bead-bound complexes used for the quantification shown in   Figure 6C . ( C ) Example images of bead-bound complexes used for the quantification shown in   Figure 6D . DOI: http://dx.doi.org/10.7554/eLife.18825.014
    Figure Legend Snippet: In vitro interaction of Nup98 and DHX9. ( A ) Anti-DHX9 antibodies coupled to beads were used to immobilize recombinant DHX9. Bead-bound DHX9 was then incubated with recombinant Nup98 or GST in the presence or absence of RNA (poly I:C), RNase A, or buffer alone. Bound proteins were analyzed by Western blots using the indicated antibodies (below images). The top row of panels shows the DHX9 bait bound to beads. The bottom row of panels shows GST and Nup98 that bound to DHX9 under the indicated conditions. Asterisks denote positions of DHX9 and Nup98. The positions of molecular mass markers (shown in kDa) are indicated on the left and right. ( B ) Example images of bead-bound complexes used for the quantification shown in Figure 6C . ( C ) Example images of bead-bound complexes used for the quantification shown in Figure 6D . DOI: http://dx.doi.org/10.7554/eLife.18825.014

    Techniques Used: In Vitro, Recombinant, Incubation, Western Blot

    22) Product Images from "Structure-Guided Mutational Analysis of a Yeast DEAD-box Protein Involved in Mitochondrial RNA Splicing"

    Article Title: Structure-Guided Mutational Analysis of a Yeast DEAD-box Protein Involved in Mitochondrial RNA Splicing

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2010.03.025

    Splicing phenotypes of mss116Δ strains expressing variants of Mss116p assessed by Northern blot analysis. (a) Schematic of the COX1 G.I, group I introns; G.II, group II introns. (b) Representative northern blots of total mitochondrial RNA from mss116Δ strains harboring mutant derivatives of Mss116p. RNA was harvested from cells grown in dextrose-containing media at 30 °C. Blots were hybridized with 5′- 32 P-labeled DNA oligonucleotides complementary to the COX1 exons aE5γ and aE6, or the intron-less COX3 transcript, which served as the loading control. Different splicing isoforms are indicated by letters A-F. Fully processed pre-mRNAs are marked as LE for ligated exons. (c) Histogram of exon ligation in each mutant strain relative to exon ligation in the wild-type strain, as detected using both the aE5γ and aE6 exon probes. All data was normalized to the abundance of the COX3 RNA. The data using the aE5γ probe are the average from two independent experiments and the error bars represent the range of the values divided by two. Exon ligation in the strains expressing the E.V., K158A, and T307A variants was very low in abundance or essentially zero and may not be seen on the histogram.
    Figure Legend Snippet: Splicing phenotypes of mss116Δ strains expressing variants of Mss116p assessed by Northern blot analysis. (a) Schematic of the COX1 G.I, group I introns; G.II, group II introns. (b) Representative northern blots of total mitochondrial RNA from mss116Δ strains harboring mutant derivatives of Mss116p. RNA was harvested from cells grown in dextrose-containing media at 30 °C. Blots were hybridized with 5′- 32 P-labeled DNA oligonucleotides complementary to the COX1 exons aE5γ and aE6, or the intron-less COX3 transcript, which served as the loading control. Different splicing isoforms are indicated by letters A-F. Fully processed pre-mRNAs are marked as LE for ligated exons. (c) Histogram of exon ligation in each mutant strain relative to exon ligation in the wild-type strain, as detected using both the aE5γ and aE6 exon probes. All data was normalized to the abundance of the COX3 RNA. The data using the aE5γ probe are the average from two independent experiments and the error bars represent the range of the values divided by two. Exon ligation in the strains expressing the E.V., K158A, and T307A variants was very low in abundance or essentially zero and may not be seen on the histogram.

    Techniques Used: Expressing, Northern Blot, Mutagenesis, Labeling, Ligation

    23) Product Images from "A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin"

    Article Title: A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.13.4667-4676.2002

    Expression of cLys domain genes in mouse macrophages. (A) cLys expression was determined by use of Competimer-normalized RT-PCR, as described in Materials and Methods. 18S mouse rRNA was used as an internal standard. Lanes 2 to 4 show results from female macrophages subjected to no selection (NS) and to 6-TG and HAT selection, respectively. Lanes 5 and 6 show male macrophages subjected to no selection and 6-TG selection, respectively. Lanes 7 to 9 show female cells identical to those in lanes 2 to 4 except for the addition of LPS treatment. Similarly, lanes 10 and 11 are male cells identical to those in lanes 5 and 6 except for the addition of LPS. Lanes 1 and 12 are control reaction mixtures containing a single set of PCR primers for either cLys (lane 1) or 18S rRNA (lane 12) only. Lane 13 is a no-template control. (B) cGas41 expression in mouse macrophages. 18S mouse rRNA was used as an internal standard. Lane M shows size markers.
    Figure Legend Snippet: Expression of cLys domain genes in mouse macrophages. (A) cLys expression was determined by use of Competimer-normalized RT-PCR, as described in Materials and Methods. 18S mouse rRNA was used as an internal standard. Lanes 2 to 4 show results from female macrophages subjected to no selection (NS) and to 6-TG and HAT selection, respectively. Lanes 5 and 6 show male macrophages subjected to no selection and 6-TG selection, respectively. Lanes 7 to 9 show female cells identical to those in lanes 2 to 4 except for the addition of LPS treatment. Similarly, lanes 10 and 11 are male cells identical to those in lanes 5 and 6 except for the addition of LPS. Lanes 1 and 12 are control reaction mixtures containing a single set of PCR primers for either cLys (lane 1) or 18S rRNA (lane 12) only. Lane 13 is a no-template control. (B) cGas41 expression in mouse macrophages. 18S mouse rRNA was used as an internal standard. Lane M shows size markers.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Selection, HAT Assay, Polymerase Chain Reaction

    24) Product Images from "Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-? and matrix metalloproteinase-13 in human chondrocytes"

    Article Title: Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-? and matrix metalloproteinase-13 in human chondrocytes

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar2700

    Human osteoarthritis chondrocytes in monolayer culture maintain their phenotype. Primary chondrocytes from osteoarthritis patients were cultured for 72 hours and were then split and cultured for an additional 3 days (passage 1). Expression of (a) type-2 collagen (COL2A1) and (b) aggrecan (ACAN), type-10 collagen (COL10A1) and SRY-box containing gene 9 (SOX-9) was determined by RT-PCR. M, 100 bp DNA ladder; P, positive control cDNA; P1, primary chondrocytes; P2, passage 1 chondrocytes.
    Figure Legend Snippet: Human osteoarthritis chondrocytes in monolayer culture maintain their phenotype. Primary chondrocytes from osteoarthritis patients were cultured for 72 hours and were then split and cultured for an additional 3 days (passage 1). Expression of (a) type-2 collagen (COL2A1) and (b) aggrecan (ACAN), type-10 collagen (COL10A1) and SRY-box containing gene 9 (SOX-9) was determined by RT-PCR. M, 100 bp DNA ladder; P, positive control cDNA; P1, primary chondrocytes; P2, passage 1 chondrocytes.

    Techniques Used: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control

    25) Product Images from "A Single Acetylation of 18 S rRNA Is Essential for Biogenesis of the Small Ribosomal Subunit in Saccharomyces cerevisiae *"

    Article Title: A Single Acetylation of 18 S rRNA Is Essential for Biogenesis of the Small Ribosomal Subunit in Saccharomyces cerevisiae *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.593996

    The 23 S pre-rRNA accumulates upon Acs2p inactivation. A , schematic depiction of acetyl-CoA metabolism in S. cerevisiae . Acetyl-CoA produced in mitochondria is used in TCA cycle for energy production. Because no ATP-citrate lyase is present in S. cerevisiae
    Figure Legend Snippet: The 23 S pre-rRNA accumulates upon Acs2p inactivation. A , schematic depiction of acetyl-CoA metabolism in S. cerevisiae . Acetyl-CoA produced in mitochondria is used in TCA cycle for energy production. Because no ATP-citrate lyase is present in S. cerevisiae

    Techniques Used: Produced

    26) Product Images from "Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes"

    Article Title: Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes

    Journal: Scientific Reports

    doi: 10.1038/srep13647

    Down-modulation of nephrin expression both at protein and transcription level in CIHPs. ( a ) Quantification (left panel) of immunoflourescence staining (right upper panel) of nephrin expression in control (Mock) and CIHPs treated with different human recombinant suPAR for 24 hours (suPAR). Results are expressed as MFI/cell and represent the average of 6 experiments ±SD. DAPI staining was used to determine nuclei number. Results are expressed as MFI/cell and represent the average of 4 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 4 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue. ( b ) Dose-dependent qPCR analysis of nephrin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. ( c ) Time course qPCR analysis of nephrin and synaptopodin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. Statistical significance ( P ) is indicated by asterisks and is represented as: ***
    Figure Legend Snippet: Down-modulation of nephrin expression both at protein and transcription level in CIHPs. ( a ) Quantification (left panel) of immunoflourescence staining (right upper panel) of nephrin expression in control (Mock) and CIHPs treated with different human recombinant suPAR for 24 hours (suPAR). Results are expressed as MFI/cell and represent the average of 6 experiments ±SD. DAPI staining was used to determine nuclei number. Results are expressed as MFI/cell and represent the average of 4 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 4 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue. ( b ) Dose-dependent qPCR analysis of nephrin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. ( c ) Time course qPCR analysis of nephrin and synaptopodin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. Statistical significance ( P ) is indicated by asterisks and is represented as: ***

    Techniques Used: Expressing, Staining, Recombinant, Real-time Polymerase Chain Reaction

    Injection of high doses of recombinant mouse suPAR into uPAR-knockout (Plaur −/− ) mouse model induces down-regulation of nephrin expression. ( a ) Quantification (left panel) of the ratio between urine total protein (mg)/creatinine (mg) concentration of suPAR treated mice with high dose of 20 μg of mouse recombinant for 24 hours vs control mice (Mock) (N = 3 mice for group). Immune-fluorescence in green (right panel) of suPAR (488 Alexa Fluor) deposit into glomerular tissue of suPAR treated Plaur −/− mice. ( b ) Quantification (left panel) of immunoflourescence staining of nephrin and synaptopodin expression in Mock and suPAR treated mice. (N = 3 mice for group). DAPI staining was used to determine cell numbers. Data are expressed as average of MFI/cell ±SD. Representative immunoflourescence staining (right panel) of nephrin in green (488 Alexa Fluor), synaptopodin in red (594 Alexa Fluor) and nucleus in blue (DAPI) expression in untreated (Mock) and suPAR treated mice (N = 3 mice for group). (c) QPCR analysis of nephrin and WT-1 expression in Mock and suPAR treated mice obtained by using specific mice TaqMan assays and expressed as relative fold change ±SD vs. mock cells. (N = 3 mice for group). Statistical significance ( P ) is indicated by asterisks and is represented as: *
    Figure Legend Snippet: Injection of high doses of recombinant mouse suPAR into uPAR-knockout (Plaur −/− ) mouse model induces down-regulation of nephrin expression. ( a ) Quantification (left panel) of the ratio between urine total protein (mg)/creatinine (mg) concentration of suPAR treated mice with high dose of 20 μg of mouse recombinant for 24 hours vs control mice (Mock) (N = 3 mice for group). Immune-fluorescence in green (right panel) of suPAR (488 Alexa Fluor) deposit into glomerular tissue of suPAR treated Plaur −/− mice. ( b ) Quantification (left panel) of immunoflourescence staining of nephrin and synaptopodin expression in Mock and suPAR treated mice. (N = 3 mice for group). DAPI staining was used to determine cell numbers. Data are expressed as average of MFI/cell ±SD. Representative immunoflourescence staining (right panel) of nephrin in green (488 Alexa Fluor), synaptopodin in red (594 Alexa Fluor) and nucleus in blue (DAPI) expression in untreated (Mock) and suPAR treated mice (N = 3 mice for group). (c) QPCR analysis of nephrin and WT-1 expression in Mock and suPAR treated mice obtained by using specific mice TaqMan assays and expressed as relative fold change ±SD vs. mock cells. (N = 3 mice for group). Statistical significance ( P ) is indicated by asterisks and is represented as: *

    Techniques Used: Injection, Recombinant, Knock-Out, Expressing, Concentration Assay, Mouse Assay, Fluorescence, Staining, Real-time Polymerase Chain Reaction

    27) Product Images from "A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium subsp. paratuberculosis in milk with the inherent possibility of differentiation between viable and dead cells"

    Article Title: A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium subsp. paratuberculosis in milk with the inherent possibility of differentiation between viable and dead cells

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-251

    Amplification plot (A) and standard curve (B) of the optimized real-time PCR assay for the Mptb52.16 target . Based on the molecular weight of the genome of MAP strain K-10 (GenBank accession number AE016958 ), 1 ng DNA equals 9.59 × 10 5 copies of the entire genome. The MAP-specific target Mptb52.16 is a single-copy gene. Thus, this figure equals the number of PCR targets per ng. The dilution series ranged from 9.59 × 10 5 to 9.59 × 10 0 copies of the genomic DNA from MAP strain CIP 103974 per PCR. Fifty copies of an IAC were added to each reaction. The MAP-specific probe was detected in the FAM channel, whereas the IAC-specific probe was detected in the HEX channel.
    Figure Legend Snippet: Amplification plot (A) and standard curve (B) of the optimized real-time PCR assay for the Mptb52.16 target . Based on the molecular weight of the genome of MAP strain K-10 (GenBank accession number AE016958 ), 1 ng DNA equals 9.59 × 10 5 copies of the entire genome. The MAP-specific target Mptb52.16 is a single-copy gene. Thus, this figure equals the number of PCR targets per ng. The dilution series ranged from 9.59 × 10 5 to 9.59 × 10 0 copies of the genomic DNA from MAP strain CIP 103974 per PCR. Fifty copies of an IAC were added to each reaction. The MAP-specific probe was detected in the FAM channel, whereas the IAC-specific probe was detected in the HEX channel.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Molecular Weight, Polymerase Chain Reaction

    28) Product Images from "Aberrant RNA methylation triggers recruitment of an alkylation repair complex"

    Article Title: Aberrant RNA methylation triggers recruitment of an alkylation repair complex

    Journal: bioRxiv

    doi: 10.1101/2020.08.28.271874

    RNA alkylation is necessary for ASCC recruitment to nuclear foci during alkylation damage. (A) In vitro demethylation assays on identical sequences of an m1A-containing RNA oligonucleotide (left) or DNA oligonucleotide (right) with wildtype or catalytically inactive (H156A) recombinant BsV-AlkB protein. Reactions were quantified by LC-MS/MS and shown as a percent of no protein control (n=4 to 5 independent replicates for each reaction; error bars indicate ± S.D. of the mean; * = p
    Figure Legend Snippet: RNA alkylation is necessary for ASCC recruitment to nuclear foci during alkylation damage. (A) In vitro demethylation assays on identical sequences of an m1A-containing RNA oligonucleotide (left) or DNA oligonucleotide (right) with wildtype or catalytically inactive (H156A) recombinant BsV-AlkB protein. Reactions were quantified by LC-MS/MS and shown as a percent of no protein control (n=4 to 5 independent replicates for each reaction; error bars indicate ± S.D. of the mean; * = p

    Techniques Used: In Vitro, Recombinant, Liquid Chromatography with Mass Spectroscopy

    29) Product Images from "MicroRNA-584 and the Protein Phosphatase and Actin Regulator 1 (PHACTR1), a New Signaling Route through Which Transforming Growth Factor-? Mediates the Migration and Actin Dynamics of Breast Cancer Cells *"

    Article Title: MicroRNA-584 and the Protein Phosphatase and Actin Regulator 1 (PHACTR1), a New Signaling Route through Which Transforming Growth Factor-? Mediates the Migration and Actin Dynamics of Breast Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.430934

    miR-584 is a novel TGF-β target. A , several cell lines were stimulated or not with 100 p m TGF-β for 24 h, and total RNA was extracted. TGF-β effect on miR-584 was evaluated by qRT-PCR in breast cancer cells of various origins.
    Figure Legend Snippet: miR-584 is a novel TGF-β target. A , several cell lines were stimulated or not with 100 p m TGF-β for 24 h, and total RNA was extracted. TGF-β effect on miR-584 was evaluated by qRT-PCR in breast cancer cells of various origins.

    Techniques Used: Quantitative RT-PCR

    PHACTR1 is regulated by miR-584. A , total RNA was extracted from MDA-MB-231 cells that were stimulated with 100 p m TGF-β for 24 h. Gene expression of several miR-584 target candidates was measured by qRT-PCR. B , SCP2 cells were transfected with
    Figure Legend Snippet: PHACTR1 is regulated by miR-584. A , total RNA was extracted from MDA-MB-231 cells that were stimulated with 100 p m TGF-β for 24 h. Gene expression of several miR-584 target candidates was measured by qRT-PCR. B , SCP2 cells were transfected with

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

    miR-584 impairs TGF-β-mediated migration. A and B , miR-584 was either overexpressed ( A ) or inhibited ( B ) with 80 n m mimic and inhibitor respectively. miR-584 levels were measured by qRT-PCR. C and D , SCP2 cells were transfected with 80 n m miR-584
    Figure Legend Snippet: miR-584 impairs TGF-β-mediated migration. A and B , miR-584 was either overexpressed ( A ) or inhibited ( B ) with 80 n m mimic and inhibitor respectively. miR-584 levels were measured by qRT-PCR. C and D , SCP2 cells were transfected with 80 n m miR-584

    Techniques Used: Migration, Quantitative RT-PCR, Transfection

    30) Product Images from "Prevention of Diabetic Nephropathy by Sulforaphane: Possible Role of Nrf2 Upregulation and Activation"

    Article Title: Prevention of Diabetic Nephropathy by Sulforaphane: Possible Role of Nrf2 Upregulation and Activation

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2012/821936

    SFN upregulated renal expression of Nrf2 and its downstream genes at protein level. Nrf2 (a) and its downstream genes, NQO1 (b), HO-1 (c), SOD1 (d), SOD2 (e), and CAT (f) expression in protein level was detected by Western blotting assay. Data were presented as means ± SD ( n = 6 at least). (* P
    Figure Legend Snippet: SFN upregulated renal expression of Nrf2 and its downstream genes at protein level. Nrf2 (a) and its downstream genes, NQO1 (b), HO-1 (c), SOD1 (d), SOD2 (e), and CAT (f) expression in protein level was detected by Western blotting assay. Data were presented as means ± SD ( n = 6 at least). (* P

    Techniques Used: Expressing, Western Blot

    SFN upregulated renal expression of Nrf2 and its downstream genes at mRNA level. Nrf2 (a) and its downstream genes, NQO1 (b), HO-1 (c), SOD1 (d), SOD2 (e), and CAT (f) expression at mRNA level were detected by RT-PCR. Data were presented as means ± SD ( n = 6 at least). (* P
    Figure Legend Snippet: SFN upregulated renal expression of Nrf2 and its downstream genes at mRNA level. Nrf2 (a) and its downstream genes, NQO1 (b), HO-1 (c), SOD1 (d), SOD2 (e), and CAT (f) expression at mRNA level were detected by RT-PCR. Data were presented as means ± SD ( n = 6 at least). (* P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    31) Product Images from "Intermittent hypoxia-induced cardiomyopathy and its prevention by Nrf2 and metallothionein"

    Article Title: Intermittent hypoxia-induced cardiomyopathy and its prevention by Nrf2 and metallothionein

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2017.07.031

    Expression of Nrf2 in response to IH exposures. ]. The NQO1 (B) and SOD2 (C) mRNA was measured by RT-PCR and Western blots. Data are presented as mean ± SD (n=5). *, p
    Figure Legend Snippet: Expression of Nrf2 in response to IH exposures. ]. The NQO1 (B) and SOD2 (C) mRNA was measured by RT-PCR and Western blots. Data are presented as mean ± SD (n=5). *, p

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

    Nrf2 and its down-stream gene expression in Nrf2-TG and WT mice exposed to 4-week IH. Expression of Nrf2 (A), NQO1 (B), and SOD2 (C) was examined by Western blots. When WB was carried out whole full membrane was separated into five horizontal strips based on Nrf2, NQO1, SOD and GAPDH molecule weights. These strips were separately incubate with each specific antibody and associated following procedures. Under this condition, therefore, all Nrf2, NQO1, and SOD2 expression were compared with the same GAPDH expression. Data are presented as mean ± SD (n=5). *, p
    Figure Legend Snippet: Nrf2 and its down-stream gene expression in Nrf2-TG and WT mice exposed to 4-week IH. Expression of Nrf2 (A), NQO1 (B), and SOD2 (C) was examined by Western blots. When WB was carried out whole full membrane was separated into five horizontal strips based on Nrf2, NQO1, SOD and GAPDH molecule weights. These strips were separately incubate with each specific antibody and associated following procedures. Under this condition, therefore, all Nrf2, NQO1, and SOD2 expression were compared with the same GAPDH expression. Data are presented as mean ± SD (n=5). *, p

    Techniques Used: Expressing, Mouse Assay, Western Blot

    Effect of PI3K inhibition on IH-induced Nrf2 expression. FVB mice were exposed to IH for 3 days with either PI3K inhibitor (LY294002) or vehicle simultaneously. The expression of p-PI3K and t-PI3K (A), p-Akt and t-Akt (B), p-GSK-3β and t-GSK-3β (C), Fyn (D), Nrf2 (E) and its target genes NQO1 (F), SOD2 (G) in heart was detected by Western blot assay and the ratio of p-PI3K and t-PI3K, p-Akt and t-Akt, p-GSK-3β and t-GSK-3β was presented. Data are presented as mean ± SD (n=5). *, p
    Figure Legend Snippet: Effect of PI3K inhibition on IH-induced Nrf2 expression. FVB mice were exposed to IH for 3 days with either PI3K inhibitor (LY294002) or vehicle simultaneously. The expression of p-PI3K and t-PI3K (A), p-Akt and t-Akt (B), p-GSK-3β and t-GSK-3β (C), Fyn (D), Nrf2 (E) and its target genes NQO1 (F), SOD2 (G) in heart was detected by Western blot assay and the ratio of p-PI3K and t-PI3K, p-Akt and t-Akt, p-GSK-3β and t-GSK-3β was presented. Data are presented as mean ± SD (n=5). *, p

    Techniques Used: Inhibition, Expressing, Mouse Assay, Western Blot

    Reciprocal regulation between Nrf2 and MT. MT-KO, MT-TG and their WT mice were exposed to IH for indicated times (A-C) and 4 weeks (D-F). Nrf2 and its target genes NQO1 and SOD2 expression was measured by Western blots. Data are presented as mean ± SD (n=5). *, p
    Figure Legend Snippet: Reciprocal regulation between Nrf2 and MT. MT-KO, MT-TG and their WT mice were exposed to IH for indicated times (A-C) and 4 weeks (D-F). Nrf2 and its target genes NQO1 and SOD2 expression was measured by Western blots. Data are presented as mean ± SD (n=5). *, p

    Techniques Used: Mouse Assay, Expressing, Western Blot

    32) Product Images from "Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma"

    Article Title: Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

    Journal: Genes to cells : devoted to molecular & cellular mechanisms

    doi: 10.1111/j.1365-2443.2010.01483.x

    Effect of overexpressing and silencing ANGPTL7/CDT6 gene on the expression of extracellular matrix (ECM) relevant proteins in primary human trabecular meshwork cells, analyzed by real-time Taq Man PCR and expressed as fold change mean ± range. (A) Fold changes of ECM relevant genes in cells transfected with ANGPTL7/CDT6 cDNA over mock negative control ( n = 3; * P ≤ 0.03). (B) Expression of ECM relevant genes in cells transfected with ANGPTL7/CDT6 siRNA over scrambled siRNA control ( n = 3; * P ≤ 0.03). Overexpression of ANGPTL7/CDT6 significantly decreases all tested ECM genes with the exception of MMP1. Silencing of ANGPTL7/CDT6 significantly increases the expression of relevant ECM genes and decreases the expression of MMP1.
    Figure Legend Snippet: Effect of overexpressing and silencing ANGPTL7/CDT6 gene on the expression of extracellular matrix (ECM) relevant proteins in primary human trabecular meshwork cells, analyzed by real-time Taq Man PCR and expressed as fold change mean ± range. (A) Fold changes of ECM relevant genes in cells transfected with ANGPTL7/CDT6 cDNA over mock negative control ( n = 3; * P ≤ 0.03). (B) Expression of ECM relevant genes in cells transfected with ANGPTL7/CDT6 siRNA over scrambled siRNA control ( n = 3; * P ≤ 0.03). Overexpression of ANGPTL7/CDT6 significantly decreases all tested ECM genes with the exception of MMP1. Silencing of ANGPTL7/CDT6 significantly increases the expression of relevant ECM genes and decreases the expression of MMP1.

    Techniques Used: Expressing, Polymerase Chain Reaction, Transfection, Negative Control, Over Expression

    Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P
    Figure Legend Snippet: Analysis of the recombinant ANGPTL7/CDT6 plasmid (pNC1) in primary human trabecular meshwork cells. Two primary HTM cell lines (HTM-55 and HTM-69) were nucleofector-transfected with either ANGPTL7/CDT6 plasmid DNA or mock-transfected, and assayed at 72-h post-transfection. (A) Normalized ANGPTL7/CDT6 cDNA in the treated cells versus the mock-transfected cells. Top panel : representative C T logarithmic curve of the hybridizations of ANGPTL7/CDT6 and endogenous 18S cDNAs from treated and mock-transfected cells with their corresponding Taq Man probes. Bottom panel : fold change of ANGPTL7/CDT6 cDNA in treated versus mock-transfected, normalized to 18S and expressed as fold change mean ± range ( n = 3 * P

    Techniques Used: Recombinant, Plasmid Preparation, Transfection

    33) Product Images from "An endogenous ribonuclease inhibitor regulates the antimicrobial activity of ribonuclease 7 in the human urinary tract"

    Article Title: An endogenous ribonuclease inhibitor regulates the antimicrobial activity of ribonuclease 7 in the human urinary tract

    Journal: Kidney international

    doi: 10.1038/ki.2013.395

    Intercalated cells express RI in the renal collecting tubule ( A ) Human kidney was labeled RNase 7 (green/arrows), aquaporin-2 (AQP-2/red), and nuclei (blue). Principal cells, identified by AQP-2 positive staining, were negative for RNase 7. ( B ) Human kidney was labeled with RNase 7 (green/arrows), RI (red/arrowheads), and nuclei (blue). RNase 7 positive cells/intercalated cells express RI. Magnification 100x.
    Figure Legend Snippet: Intercalated cells express RI in the renal collecting tubule ( A ) Human kidney was labeled RNase 7 (green/arrows), aquaporin-2 (AQP-2/red), and nuclei (blue). Principal cells, identified by AQP-2 positive staining, were negative for RNase 7. ( B ) Human kidney was labeled with RNase 7 (green/arrows), RI (red/arrowheads), and nuclei (blue). RNase 7 positive cells/intercalated cells express RI. Magnification 100x.

    Techniques Used: Labeling, Staining

    RI binds RNase 7 ( A ) Binding of recombinant RNase 7 to RI was confirmed by native gel electrophoresis. Representative native gels demonstrate a mobility shift in recombinant RI upon RNase 7 binding as visualized by silver-staining. Results also show that the interaction of RI with RNase 7 can be blocked by pre-incubation with the RI inhibitor p -HMB. ( B ) Binding of endogenous RNase 7 to RI was confirmed using co-immunoprecipitation assays. Lysates of human bladder (B), non-infected kidney tissue (Nl), and kidney tissue with pyelonephritis (P) were immunoprecipitated with anti-RI monoclonal antibody, subjected to SDS-PAGE, followed by Western immunoblot analysis using anti-RNase 7 polyclonal antibody. Results indicate that RNase 7 complexes with RI in human bladder and kidney tissue. 25ng recombinant RNase 7 served as control. ( C ) Urinary RI from non-infected urine (NI) and urine infected with E. coli (I) was pulled down using recombinant RNase 7 and subjected to SDS-PAGE followed by Western immunoblot analysis using anti-RI monoclonal antibody. Results demonstrate that urinary RI in infected urine binds recombinant RNase 7. 50ng recombinant RI served as control.
    Figure Legend Snippet: RI binds RNase 7 ( A ) Binding of recombinant RNase 7 to RI was confirmed by native gel electrophoresis. Representative native gels demonstrate a mobility shift in recombinant RI upon RNase 7 binding as visualized by silver-staining. Results also show that the interaction of RI with RNase 7 can be blocked by pre-incubation with the RI inhibitor p -HMB. ( B ) Binding of endogenous RNase 7 to RI was confirmed using co-immunoprecipitation assays. Lysates of human bladder (B), non-infected kidney tissue (Nl), and kidney tissue with pyelonephritis (P) were immunoprecipitated with anti-RI monoclonal antibody, subjected to SDS-PAGE, followed by Western immunoblot analysis using anti-RNase 7 polyclonal antibody. Results indicate that RNase 7 complexes with RI in human bladder and kidney tissue. 25ng recombinant RNase 7 served as control. ( C ) Urinary RI from non-infected urine (NI) and urine infected with E. coli (I) was pulled down using recombinant RNase 7 and subjected to SDS-PAGE followed by Western immunoblot analysis using anti-RI monoclonal antibody. Results demonstrate that urinary RI in infected urine binds recombinant RNase 7. 50ng recombinant RI served as control.

    Techniques Used: Binding Assay, Recombinant, Nucleic Acid Electrophoresis, Mobility Shift, Silver Staining, Incubation, Immunoprecipitation, Infection, SDS Page, Western Blot

    RI blocks RNase 7 binding to lipopolysaccharide ( A ) To determine if RI alters RNase 7 bacterial binding, E. coli were incubated with 2 μM RNase 7 (R7), RNase 7 pre-incubated with RI (R7/RI), or 2 μM RI alone. After centrifugation, the supernatant and pellet fraction were subjected to SDS-PAGE and visualized by Coomassie Blue staining. Supernatant represents the soluble fraction that contains unbound protein while the pellet fraction contains the E.coli -bound peptides. Results demonstrate that RNase 7 binds uropathogenic E. coli (E) and that binding is reduced in the presence of RI. ( B ) Displacement of LPS-bound Bodipy TR cadavarine with increasing concentrations of RNase 7, Polymyxin B, RNase A, and RI. Results confirm that RNase 7 binds LPS while RI does not. ( C ) Displacement of LPS-bound Bodipy TR cadavarine with increasing concentrations of RI pre-incubated with 1 μM RNase 7. Results confirm that the addition of RI reduces RNase 7 binding to LPS. The addition of p -HMB to RI improves RNase 7 binding to LPS.
    Figure Legend Snippet: RI blocks RNase 7 binding to lipopolysaccharide ( A ) To determine if RI alters RNase 7 bacterial binding, E. coli were incubated with 2 μM RNase 7 (R7), RNase 7 pre-incubated with RI (R7/RI), or 2 μM RI alone. After centrifugation, the supernatant and pellet fraction were subjected to SDS-PAGE and visualized by Coomassie Blue staining. Supernatant represents the soluble fraction that contains unbound protein while the pellet fraction contains the E.coli -bound peptides. Results demonstrate that RNase 7 binds uropathogenic E. coli (E) and that binding is reduced in the presence of RI. ( B ) Displacement of LPS-bound Bodipy TR cadavarine with increasing concentrations of RNase 7, Polymyxin B, RNase A, and RI. Results confirm that RNase 7 binds LPS while RI does not. ( C ) Displacement of LPS-bound Bodipy TR cadavarine with increasing concentrations of RI pre-incubated with 1 μM RNase 7. Results confirm that the addition of RI reduces RNase 7 binding to LPS. The addition of p -HMB to RI improves RNase 7 binding to LPS.

    Techniques Used: Binding Assay, Incubation, Centrifugation, SDS Page, Staining

    Neutrophil proteases degrade RI ( A ) Representative silver stained SDS-PAGE gels demonstrating recombinant RI proteolysis by the neutrophil proteases neutrophil elastase (NE) and proteinase 3 (Pr3). Lane 1: molecular weight marker (MW), Lane 2: 3 μg recombinant RI without proteases, Lane 3: RI incubated with 100ng NE, Lane 4–6: recombinant RI incubated with 6ng, 30ng, and 60ng Pr3, respectively. ( B/C ) Clinical urine isolates infected with E. coli were incubated with and without protease inhibitor cocktail (PI) and subjected to SDS-PAGE followed by Western immunoblot analysis using a monoclonal antibody directed against RI or a polyclonal antibody directed against RNase 7. (B) Results demonstrate that urine proteases degrade urinary RI and protease inhibitor cocktail prevents RI proteolysis. (C) Urinary proteases did not cause significant RNase 7 degradation. 50ng recombinant RI or RNase 7 served as control.
    Figure Legend Snippet: Neutrophil proteases degrade RI ( A ) Representative silver stained SDS-PAGE gels demonstrating recombinant RI proteolysis by the neutrophil proteases neutrophil elastase (NE) and proteinase 3 (Pr3). Lane 1: molecular weight marker (MW), Lane 2: 3 μg recombinant RI without proteases, Lane 3: RI incubated with 100ng NE, Lane 4–6: recombinant RI incubated with 6ng, 30ng, and 60ng Pr3, respectively. ( B/C ) Clinical urine isolates infected with E. coli were incubated with and without protease inhibitor cocktail (PI) and subjected to SDS-PAGE followed by Western immunoblot analysis using a monoclonal antibody directed against RI or a polyclonal antibody directed against RNase 7. (B) Results demonstrate that urine proteases degrade urinary RI and protease inhibitor cocktail prevents RI proteolysis. (C) Urinary proteases did not cause significant RNase 7 degradation. 50ng recombinant RI or RNase 7 served as control.

    Techniques Used: Staining, SDS Page, Recombinant, Molecular Weight, Marker, Incubation, Infection, Protease Inhibitor, Western Blot

    RI inhibits the antimicrobial activity of RNase 7 against E. coli ( A ) Uropathogenic E. coli was exposed to 2 μM RNase 7, equal concentrations of recombinant RNase 7 pre-incubated with RI (RNase 7-RI), or 2 μM of RI. Untreated bacteria served as the control. The results are displayed as the percentage of remaining CFUs in relation to untreated controls. The antimicrobial activity of RNase 7 was significantly reduced in the presence of RI. Data represent the mean of triplicates ± SEM. ( B ) E. coli were stained using a 1:1 mixture of SYTO9 and propidium iodide. The SYTO9-stained cells (green) represent live cells and the propidium iodide-stained cells (red) represent killed cells. Bacterial viability was visualized after exposure to RNase 7 with and without RI at 180 minutes. Magnification 63x. (C) E. coli were stained using a 1:1 mixture of SYTO9 and propidium iodide and incubated with 2 μM RNase 7, equal concentrations of recombinant RNase 7 pre-incubated with RI (RNase 7-RI), or 2 μM of RI. Bacterial viability over time was analyzed integrating fluorescent changes in SYTO9 and propidium iodide dye. Values are the average of three replicates.
    Figure Legend Snippet: RI inhibits the antimicrobial activity of RNase 7 against E. coli ( A ) Uropathogenic E. coli was exposed to 2 μM RNase 7, equal concentrations of recombinant RNase 7 pre-incubated with RI (RNase 7-RI), or 2 μM of RI. Untreated bacteria served as the control. The results are displayed as the percentage of remaining CFUs in relation to untreated controls. The antimicrobial activity of RNase 7 was significantly reduced in the presence of RI. Data represent the mean of triplicates ± SEM. ( B ) E. coli were stained using a 1:1 mixture of SYTO9 and propidium iodide. The SYTO9-stained cells (green) represent live cells and the propidium iodide-stained cells (red) represent killed cells. Bacterial viability was visualized after exposure to RNase 7 with and without RI at 180 minutes. Magnification 63x. (C) E. coli were stained using a 1:1 mixture of SYTO9 and propidium iodide and incubated with 2 μM RNase 7, equal concentrations of recombinant RNase 7 pre-incubated with RI (RNase 7-RI), or 2 μM of RI. Bacterial viability over time was analyzed integrating fluorescent changes in SYTO9 and propidium iodide dye. Values are the average of three replicates.

    Techniques Used: Activity Assay, Recombinant, Incubation, Staining

    Kidney RI and RNase 7 peptide production during sterility and pyelonephritis To confirm the mRNA expression of RNH1 and RNASE7 pattern is accompanied by alterations in peptide production, ELISA quantitated RI and RNase 7 peptide from the same non-infected kidney tissue and kidney tissue with pyelonephritis used in real-time PCR analysis. RI production significantly decreased with pyelonephritis ( p =0.0068) while RNase 7 expression significantly increased ( p =0.0386).
    Figure Legend Snippet: Kidney RI and RNase 7 peptide production during sterility and pyelonephritis To confirm the mRNA expression of RNH1 and RNASE7 pattern is accompanied by alterations in peptide production, ELISA quantitated RI and RNase 7 peptide from the same non-infected kidney tissue and kidney tissue with pyelonephritis used in real-time PCR analysis. RI production significantly decreased with pyelonephritis ( p =0.0068) while RNase 7 expression significantly increased ( p =0.0386).

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

    RI neutralizes the antimicrobial activity of urinary RNase 7 The antimicrobial properties of urinary RNase 7 were measured as changes in turbidity of cultured human urine using the absorbance at 600 nm (OD 600 ). Human urine samples were inoculated with E. coli (PEDUTI-89 or CFT073) as shown by the dashed line. Addition of RI (solid black line) or RNase 7 monoclonal antibody (diamond studded line) blocked the antimicrobial activity of RNase 7, resulting in increased bacterial growth. The open circles represent non-inoculated urine samples.
    Figure Legend Snippet: RI neutralizes the antimicrobial activity of urinary RNase 7 The antimicrobial properties of urinary RNase 7 were measured as changes in turbidity of cultured human urine using the absorbance at 600 nm (OD 600 ). Human urine samples were inoculated with E. coli (PEDUTI-89 or CFT073) as shown by the dashed line. Addition of RI (solid black line) or RNase 7 monoclonal antibody (diamond studded line) blocked the antimicrobial activity of RNase 7, resulting in increased bacterial growth. The open circles represent non-inoculated urine samples.

    Techniques Used: Activity Assay, Cell Culture

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

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

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00096

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

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

    Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.
    Figure Legend Snippet: Expression of HsAGO2 generates morphological defects in Arabidopsis. (A) 22-day old primary transformants for the 35S:HsAGO2 construct were categorized based on the apparent severity of their morphological phenotypes. Increased leaf serration distinguished “mild” phenotypes from wild type (WT), “obvious” phenotypes were characterized by broadened leaves, serration, accelerated senescence and some upward leaf-curl, while phenotypes considered “severe” were distinguished by strong upward leaf-curl in addition. Scale bars represent 10 mm. (B) The abundances of HsAGO2 mRNA was measured in total RNA from sample pools composed of 4–8 transformants, 22-days old, from each morphological category. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. All measurements are normalized to CYCLOPHILIN mRNA. Data is averaged from three technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean.

    Techniques Used: Expressing, Construct

    miRNA abundances decrease in AGO overexpressing plants. The abundances of miR159a and miR166 were measured in total RNA from sample pools composed of 4–8 transformants, 24-days old, from each morphological category for each construct. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. miRNA levels are normalized to the small RNA sno101. Data is averaged from two technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean. Values marked with * are significantly smaller ( P
    Figure Legend Snippet: miRNA abundances decrease in AGO overexpressing plants. The abundances of miR159a and miR166 were measured in total RNA from sample pools composed of 4–8 transformants, 24-days old, from each morphological category for each construct. Wild type (WT) and ago1–27 plants, grown in parallel, were included as controls. miRNA levels are normalized to the small RNA sno101. Data is averaged from two technical cDNA replicates, each of which comprised triplicate measurements, and error bars depict standard error of the mean. Values marked with * are significantly smaller ( P

    Techniques Used: Construct

    35) Product Images from "Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer, et al. Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer"

    Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer, et al. Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

    Journal: Cancer Science

    doi: 10.1111/cas.13467

    15‐Hydroxyprostaglandin dehydrogenase (15‐ PGDH ) downregulation by interleukin‐1β ( IL ‐1β) enhances pancreatic ductal adenocarcinoma cell growth. A,B, Expression of HPGD (the gene coding 15‐ PGDH protein, upper panel) or 15‐ PGDH (lower panel) in PK ‐8 cells (A) or S2‐013 cells (B) after treatment with si RNA targeting 15‐ PGDH or with control si RNA , evaluated by quantitative RT ‐ PCR (upper panel) or Western blot analysis (lower panel). Data are presented as the treated/control cell ratio. C,D, PK ‐8 cells (C) or S2‐013 cells (D) transfected with si RNA s targeting 15‐ PGDH or with control si RNA were incubated for up to 96 hours and assayed for cell number; data are presented as the treated/control (time = 0) cell ratio. E,F, Expression of 15‐ PGDH in PK ‐8 cells or S2‐013 cells after IL ‐1β (E) or tumor necrosis factor‐α ( TNF ‐α) (F) treatment for 24 and 48 hours and distilled water treatment for 48 hours as a control was evaluated by Western blotting. G, Column graph showing relative 15‐ PGDH levels in PK ‐8 cells or S2‐013 cells after IL ‐1β and TNF ‐α treatment for 24 and 48 hours, and distilled water treatment for 48 hours as a control, were evaluated using ImageJ software. H, Expression of HPGD and IL 1B in six PDAC patients determined by quantitative RT ‐ PCR . Data were normalized to the ACTB mRNA level and are shown as the mean ± SD of three independent experiments. **P
    Figure Legend Snippet: 15‐Hydroxyprostaglandin dehydrogenase (15‐ PGDH ) downregulation by interleukin‐1β ( IL ‐1β) enhances pancreatic ductal adenocarcinoma cell growth. A,B, Expression of HPGD (the gene coding 15‐ PGDH protein, upper panel) or 15‐ PGDH (lower panel) in PK ‐8 cells (A) or S2‐013 cells (B) after treatment with si RNA targeting 15‐ PGDH or with control si RNA , evaluated by quantitative RT ‐ PCR (upper panel) or Western blot analysis (lower panel). Data are presented as the treated/control cell ratio. C,D, PK ‐8 cells (C) or S2‐013 cells (D) transfected with si RNA s targeting 15‐ PGDH or with control si RNA were incubated for up to 96 hours and assayed for cell number; data are presented as the treated/control (time = 0) cell ratio. E,F, Expression of 15‐ PGDH in PK ‐8 cells or S2‐013 cells after IL ‐1β (E) or tumor necrosis factor‐α ( TNF ‐α) (F) treatment for 24 and 48 hours and distilled water treatment for 48 hours as a control was evaluated by Western blotting. G, Column graph showing relative 15‐ PGDH levels in PK ‐8 cells or S2‐013 cells after IL ‐1β and TNF ‐α treatment for 24 and 48 hours, and distilled water treatment for 48 hours as a control, were evaluated using ImageJ software. H, Expression of HPGD and IL 1B in six PDAC patients determined by quantitative RT ‐ PCR . Data were normalized to the ACTB mRNA level and are shown as the mean ± SD of three independent experiments. **P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Incubation, Software

    36) Product Images from "Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)"

    Article Title: Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA)

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt634

    Expression, localization and tRNA Asp(GUC) methylation activity of DnmA under various conditions. ( A ) dnmA expression is regulated during the cell cycle. Cells were arrested in the cell cycle by cold treatment and then released by transfer to 22°C. Cells were counted every 30 min (grey dots and grey line), and samples were taken for qPCR (black bars). After cell division, dnmA expression increased ∼5-fold and then rapidly declined to basal levels. Normalization was done on vegetative growing cells. dnmA expression is only shown from 3 to 4.5 h during recovery. ( B ) DnmA is lost from the nucleus during mitosis (arrow). The three cells on the right are in S-phase as indicated by the RFP-PCNA marker and accumulate DnmA in the nucleus (for further details see movie in Supplementary Material ). ( C ) Relative quantification of dnmA expression levels in AX2 cells after 2.5 h recovery from cold shock at 4°C. Expression of dnmA increased > 40-fold and returned within 30 min to basal levels (n = 3). ( D ) At 16 h of development, dnmA expression increased ∼46-fold in the NC4 strain. In AX2 cells, expression increased only ∼5-fold. ( E ) At 16 h of development, in vivo methylation of C38 in tRNA Asp(GUC) increased up to 75% in the D. discoideum strain NC4, whereas no significant increase was observed in AX2 cells.
    Figure Legend Snippet: Expression, localization and tRNA Asp(GUC) methylation activity of DnmA under various conditions. ( A ) dnmA expression is regulated during the cell cycle. Cells were arrested in the cell cycle by cold treatment and then released by transfer to 22°C. Cells were counted every 30 min (grey dots and grey line), and samples were taken for qPCR (black bars). After cell division, dnmA expression increased ∼5-fold and then rapidly declined to basal levels. Normalization was done on vegetative growing cells. dnmA expression is only shown from 3 to 4.5 h during recovery. ( B ) DnmA is lost from the nucleus during mitosis (arrow). The three cells on the right are in S-phase as indicated by the RFP-PCNA marker and accumulate DnmA in the nucleus (for further details see movie in Supplementary Material ). ( C ) Relative quantification of dnmA expression levels in AX2 cells after 2.5 h recovery from cold shock at 4°C. Expression of dnmA increased > 40-fold and returned within 30 min to basal levels (n = 3). ( D ) At 16 h of development, dnmA expression increased ∼46-fold in the NC4 strain. In AX2 cells, expression increased only ∼5-fold. ( E ) At 16 h of development, in vivo methylation of C38 in tRNA Asp(GUC) increased up to 75% in the D. discoideum strain NC4, whereas no significant increase was observed in AX2 cells.

    Techniques Used: Expressing, Methylation, Activity Assay, Real-time Polymerase Chain Reaction, Marker, In Vivo

    In vitro methylation of tRNA Gly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3 H-labelled methylated tRNAs. As a control tRNA Asp(GUC) methylation is shown.
    Figure Legend Snippet: In vitro methylation of tRNA Gly(GCC) by recombinant DnmA and hDnmt2. In vitro transcribed tRNAs were methylated in vitro as described before. The top panel shows the ethidium-bromide stained gel, the bottom panel the fluorogram of 3 H-labelled methylated tRNAs. As a control tRNA Asp(GUC) methylation is shown.

    Techniques Used: In Vitro, Methylation, Recombinant, Staining

    Ex vivo methylation and blocking assay. ( A ) Methylation of in vitro transcribed tRNA Asp(GUC) was completely blocked when a complementary antisense oligo was hybridized. ( B ) Using enriched small RNA from dnmA KO cells, ex vivo methylation in the tRNA size class was differentially lost when antisense oligos to tRNA Asp(GUC) and tRNA Glu(UUC) were hybridized before the methylation reaction. The oligo against tRNA Glu(UUC) also covers tRNA Glu(CUC) with minor mismatches (see Supplementary Figure S2 ). Even with both oligos, a significant amount of 3 H incorporation still remained. The upper panel shows the ethidiumbromide stained gel to demonstrate equal loading, the lower panel shows the fluorogram. The arrow marks a band that was specifically lost when the anti tRNA Glu(UUC) oligo was used.
    Figure Legend Snippet: Ex vivo methylation and blocking assay. ( A ) Methylation of in vitro transcribed tRNA Asp(GUC) was completely blocked when a complementary antisense oligo was hybridized. ( B ) Using enriched small RNA from dnmA KO cells, ex vivo methylation in the tRNA size class was differentially lost when antisense oligos to tRNA Asp(GUC) and tRNA Glu(UUC) were hybridized before the methylation reaction. The oligo against tRNA Glu(UUC) also covers tRNA Glu(CUC) with minor mismatches (see Supplementary Figure S2 ). Even with both oligos, a significant amount of 3 H incorporation still remained. The upper panel shows the ethidiumbromide stained gel to demonstrate equal loading, the lower panel shows the fluorogram. The arrow marks a band that was specifically lost when the anti tRNA Glu(UUC) oligo was used.

    Techniques Used: Ex Vivo, Methylation, Blocking Assay, In Vitro, Staining

    Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. ( A ) Examples of time courses on covalent complex formation with tRNA Asp(GUC) and tRNA Glu(UUC) . ( B ) Complex bands in A were quantified and presented on a time scale.
    Figure Legend Snippet: Formation and turnover of tRNA-methyltransferase complexes. Turnover of covalent complexes of tRNA substrates with DnmA and hDnmt2. ( A ) Examples of time courses on covalent complex formation with tRNA Asp(GUC) and tRNA Glu(UUC) . ( B ) Complex bands in A were quantified and presented on a time scale.

    Techniques Used:

    tRNA and tRNA fragments associated with DnmA-GFP. After UV crosslinking, RNA bound to DnmA was co-immunoprecipitated under denaturing conditions (CLIP). The number of normalised reads for tRNAs detected by Illumina sequencing is shown for DnmA CLIP and for the control CLIP with GFP. In addition to full-length tRNAs, four classes of tRNA fragments were found, and these are indicated by different shading of the bars. Size and localization of fragments is shown schematically on the simplified clover leaf structure. tRNAs that are not significantly enriched with DnmA are shown in Supplementary Figure S6 . In case of multiple gene copys resulting in the same RNA transcript, the sequencing reads were mapped to the first copy in the genome starting from chromsome 1. Only this copy is listed. Sequences and detailed information on gene copies and potential isoacceptors are listed in Supplementary Table S2 .
    Figure Legend Snippet: tRNA and tRNA fragments associated with DnmA-GFP. After UV crosslinking, RNA bound to DnmA was co-immunoprecipitated under denaturing conditions (CLIP). The number of normalised reads for tRNAs detected by Illumina sequencing is shown for DnmA CLIP and for the control CLIP with GFP. In addition to full-length tRNAs, four classes of tRNA fragments were found, and these are indicated by different shading of the bars. Size and localization of fragments is shown schematically on the simplified clover leaf structure. tRNAs that are not significantly enriched with DnmA are shown in Supplementary Figure S6 . In case of multiple gene copys resulting in the same RNA transcript, the sequencing reads were mapped to the first copy in the genome starting from chromsome 1. Only this copy is listed. Sequences and detailed information on gene copies and potential isoacceptors are listed in Supplementary Table S2 .

    Techniques Used: Immunoprecipitation, Cross-linking Immunoprecipitation, Sequencing

    In vitro methylation of tRNA Asp(GUC) . ( A ) In vitro methylation of tRNA Asp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl 2 and by hDnmt2 at 5 mM Mg 2+ . The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3 H-Me in the tRNAs. Reactions were run for the times indicated. ( B ) In vivo methylation of cytosines in tRNA Asp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNA Asp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. ( C ) In vitro methylation of small enriched RNA of a dnmA KO strain ( ex vivo methylation). The methylation reaction was done for the times indicated.
    Figure Legend Snippet: In vitro methylation of tRNA Asp(GUC) . ( A ) In vitro methylation of tRNA Asp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl 2 and by hDnmt2 at 5 mM Mg 2+ . The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3 H-Me in the tRNAs. Reactions were run for the times indicated. ( B ) In vivo methylation of cytosines in tRNA Asp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNA Asp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. ( C ) In vitro methylation of small enriched RNA of a dnmA KO strain ( ex vivo methylation). The methylation reaction was done for the times indicated.

    Techniques Used: In Vitro, Methylation, Staining, In Vivo, Methylation Sequencing, Sequencing, Ex Vivo

    37) Product Images from "The Aspergillus nidulans Zn(II)2Cys6 transcription factor AN5673/RhaR mediates L-rhamnose utilization and the production of α-L-rhamnosidases"

    Article Title: The Aspergillus nidulans Zn(II)2Cys6 transcription factor AN5673/RhaR mediates L-rhamnose utilization and the production of α-L-rhamnosidases

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-014-0161-9

    Extracellular α-L-rhamnosidase activity and α-L-rhamnosidase gene expression in rhaR + and ∆ rhaR strains. (A) Bar diagram of the relative α-L-rhamnosidase activities secreted by rhaR + (AR4, AR70, AR198, AR271 and AR256) and ∆ rhaR (AR225, AR227, AR237 and AR234) strains after 48 h grown in 1% w/v L-rhamnose. Activities are presented as percentages of those observed in AR198, and values are presented as the mean of at least three independent experiments and their standard deviation. (B) RT-PCR analyses for rhaA and rhaE in rhaR + and ∆ rhaR strains under inducing conditions using RNAs isolated from mycelia obtained after 4 h transfer to 1% w/v L-rhamnose. The actin actA gene (AN6542) was used as a constitutive control for normalization. RNA quality and amount was also verified by ethidium bromide staining of rRNAs (not shown).
    Figure Legend Snippet: Extracellular α-L-rhamnosidase activity and α-L-rhamnosidase gene expression in rhaR + and ∆ rhaR strains. (A) Bar diagram of the relative α-L-rhamnosidase activities secreted by rhaR + (AR4, AR70, AR198, AR271 and AR256) and ∆ rhaR (AR225, AR227, AR237 and AR234) strains after 48 h grown in 1% w/v L-rhamnose. Activities are presented as percentages of those observed in AR198, and values are presented as the mean of at least three independent experiments and their standard deviation. (B) RT-PCR analyses for rhaA and rhaE in rhaR + and ∆ rhaR strains under inducing conditions using RNAs isolated from mycelia obtained after 4 h transfer to 1% w/v L-rhamnose. The actin actA gene (AN6542) was used as a constitutive control for normalization. RNA quality and amount was also verified by ethidium bromide staining of rRNAs (not shown).

    Techniques Used: Activity Assay, Expressing, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Isolation, Staining

    38) Product Images from "MiR-663a Stimulates Proliferation and Suppresses Early Apoptosis of Human Spermatogonial Stem Cells by Targeting NFIX and Regulating Cell Cycle"

    Article Title: MiR-663a Stimulates Proliferation and Suppresses Early Apoptosis of Human Spermatogonial Stem Cells by Targeting NFIX and Regulating Cell Cycle

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.015

    Identification of the Human SSC Line (A and B) RT-PCR showed the mRNA levels of VASA , MEGEA4 , THY1 , RET , GPR125 , PLZF , UCHL1 , and GFRA1 in the human SSC line (A) and testicular tissues of OA patients (B, positive control). Samples without cDNA (no cDNA) but PCR with gene primers were used as negative controls, and GAPDH served as a loading control of total RNA. (C) Immunocytochemistry of anti-MAGEA4 stained by diaminobenzine (DAB) showed the presence of MAGEA4 protein (left panel) in the human SSC line. Replacement of anti-MAGEA4 with isotype IgGs was used as a negative control (right panel). (D–I) Immunofluorescence demonstrated the expression of VASA (D), UCHL1 (E), GFRA1 (F), GPR125 (G), THY1 (H), and SV40 (I) in the human SSC line. (J) Normal IgG was substituted for primary antibodies as a negative control. Scale bars, 10 μm (C–J).
    Figure Legend Snippet: Identification of the Human SSC Line (A and B) RT-PCR showed the mRNA levels of VASA , MEGEA4 , THY1 , RET , GPR125 , PLZF , UCHL1 , and GFRA1 in the human SSC line (A) and testicular tissues of OA patients (B, positive control). Samples without cDNA (no cDNA) but PCR with gene primers were used as negative controls, and GAPDH served as a loading control of total RNA. (C) Immunocytochemistry of anti-MAGEA4 stained by diaminobenzine (DAB) showed the presence of MAGEA4 protein (left panel) in the human SSC line. Replacement of anti-MAGEA4 with isotype IgGs was used as a negative control (right panel). (D–I) Immunofluorescence demonstrated the expression of VASA (D), UCHL1 (E), GFRA1 (F), GPR125 (G), THY1 (H), and SV40 (I) in the human SSC line. (J) Normal IgG was substituted for primary antibodies as a negative control. Scale bars, 10 μm (C–J).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Immunocytochemistry, Staining, Negative Control, Immunofluorescence, Expressing

    39) Product Images from "Deletion of Metallothionein Exacerbates Intermittent Hypoxia-Induced Oxidative and Inflammatory Injury in Aorta"

    Article Title: Deletion of Metallothionein Exacerbates Intermittent Hypoxia-Induced Oxidative and Inflammatory Injury in Aorta

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2014/141053

    MT-KO mice exhibited earlier and more severe IH-induced aortic inflammation. Aortic inflammation was examined by immunohistochemical staining and qRT-PCR for the expression of TNF- α (a) and immunohistochemical staining for VCAM-1 (b), followed by semiquantitative analysis. Data were presented as means ± SDs ( n = 6); * P
    Figure Legend Snippet: MT-KO mice exhibited earlier and more severe IH-induced aortic inflammation. Aortic inflammation was examined by immunohistochemical staining and qRT-PCR for the expression of TNF- α (a) and immunohistochemical staining for VCAM-1 (b), followed by semiquantitative analysis. Data were presented as means ± SDs ( n = 6); * P

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

    40) Product Images from "Transforming growth factor β-induced epithelial to mesenchymal transition requires the Ste20-like kinase SLK independently of its catalytic activity"

    Article Title: Transforming growth factor β-induced epithelial to mesenchymal transition requires the Ste20-like kinase SLK independently of its catalytic activity

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21928

    SLK knockdown significantly inhibits Snai1 and vimentin expression following TGFβ1 treatment (A) NMuMG cells were infected with either AdshScrambled or AdshSLK for 48 hours. The cultures were stimulated with TGFβ and with and surveyed for SLK and vimentin expression. (B) Total RNA was extracted from identical cultures and Snai1 (B) and E-Cadherin (C) mRNA levels were monitored by Q-PCR. Normalization was performed against GAPDH mRNA levels. Each experiment was run in triplicate with three biological replicates. * p
    Figure Legend Snippet: SLK knockdown significantly inhibits Snai1 and vimentin expression following TGFβ1 treatment (A) NMuMG cells were infected with either AdshScrambled or AdshSLK for 48 hours. The cultures were stimulated with TGFβ and with and surveyed for SLK and vimentin expression. (B) Total RNA was extracted from identical cultures and Snai1 (B) and E-Cadherin (C) mRNA levels were monitored by Q-PCR. Normalization was performed against GAPDH mRNA levels. Each experiment was run in triplicate with three biological replicates. * p

    Techniques Used: Expressing, Infection, Polymerase Chain Reaction

    SLK regulates EMT independently of its kinase activity (A) NMuMG cells were treated with 2ng/mL of TGFβ1 for various times and SLK was immunoprecipitated and subjected to in vitro kinase assays. IP= immunoprecipitate, IB= immunoblot, WCL= whole cell lysate. (B) NMuMG cells were transfected with a wildtype or dominant negative (K63R) SLK construct and total SLK was immunoprecipitated and assayed for kinase activity. IB= immunoblot. Total RNA was also extracted from the transfected cultures following TGFβ stimulation (2ng/ml for 9 hours) and assayed for SLK (C) or Snai1 (D) expression. mRNA levels were normalized to GAPDH and directly compared to AdshSLK-infected cultures. Each experiment was run in triplicate with three biological replicates. Error bars represent the standard error. * p
    Figure Legend Snippet: SLK regulates EMT independently of its kinase activity (A) NMuMG cells were treated with 2ng/mL of TGFβ1 for various times and SLK was immunoprecipitated and subjected to in vitro kinase assays. IP= immunoprecipitate, IB= immunoblot, WCL= whole cell lysate. (B) NMuMG cells were transfected with a wildtype or dominant negative (K63R) SLK construct and total SLK was immunoprecipitated and assayed for kinase activity. IB= immunoblot. Total RNA was also extracted from the transfected cultures following TGFβ stimulation (2ng/ml for 9 hours) and assayed for SLK (C) or Snai1 (D) expression. mRNA levels were normalized to GAPDH and directly compared to AdshSLK-infected cultures. Each experiment was run in triplicate with three biological replicates. Error bars represent the standard error. * p

    Techniques Used: Activity Assay, Immunoprecipitation, In Vitro, Transfection, Dominant Negative Mutation, Construct, Expressing, Infection

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