click-it nascent rna capture kit Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher click it rna alexa fluor 488 imaging kit
    EVs contain plasmodial RNAs. ( a ) Quantification of P. falciparum RNAs transferred to EVs. Infected RBCs were labeled with Click-It <t>RNA</t> <t>Alexa</t> Fluor 488. After 30 h. of incubation, EVs were collected and EU incorporation was analyzed by Flow Cytometry. ( b ) Distribution of the reads mapped to the P. falciparum genome according to the corresponding chromosomes. ( c ) Distribution of the reads mapped to the P. falciparum according to the size of the chromosome. ( d ) Types of Plasmodium genes in the EVs by RNA-Seq. ( e ) Extracellular vesicles contain a large number of Apicoplast tRNA and Mitochondrial rRNA. ( f ) RNA expression is different in EVs than mixed stage parasite expression pattern. ( g ) EV RNA composition is different that of iRBC. The expression levels shown by RNAseq of EV has no detectable correlation with that of steady state iRBC, showing that EV is not merely a smaller form of iRBC. Neither parametric (Pearson’s R) nor parametric correlations (Kendell’s tau) analysis detected correlation between EV and iRBC at any time points with correlation larger than 0.001. The X-axis shows the average steady state RNA expression levels.
    Click It Rna Alexa Fluor 488 Imaging Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 532 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/click it rna alexa fluor 488 imaging kit/product/Thermo Fisher
    Average 99 stars, based on 532 article reviews
    Price from $9.99 to $1999.99
    click it rna alexa fluor 488 imaging kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Thermo Fisher click it nascent rna capture kit
    <t>FOXC2-AS1</t> positively regulated FOXC2 expression via enhancing FOXC2 mRNA stability. a The relative expression of FOXC2-AS1 in the cytoplasm and nucleus of SW480 and LoVo cells. FISH detection was used to investigate the location of FOXC2-AS1 in cells ( b ) and tissues ( c ). Scale bars = 100 μm. d The expression of FOXC2 was examined in 66 CRC and 15 adjacent normal tissues. e The expression relationship between FOXC2-AS1 and FOXC2 was analyzed in 66 CRC tissues. FOXC2 expression was examined in FOXC2-AS1-silenced SW480 and LoVo cells by qRT-PCR method ( f ) and Western blot ( g ). h qRT-PCR detected the levels of nascent FOXC2 pre-mRNA with Click-iT Nascent <t>RNA</t> Capture Kit in FOXC2-AS1-silenced and control cells. i qRT-PCR investigated FOXC2 mRNA stability in FOXC2-AS1-silenced and control cells treated with the transcriptional inhibitor actinomycin D (50 ng/ml) for different times. j Schematic diagram of FOXC2-AS1 and FOXC2 gene locus and structure. The red grid represents the completely complementary region. The number represents the length of exon or intron. k qRT-PCR was conducted to analyze RNase protection experiment. β-actin was used as a negative control, while PDCD4-AS1 was used as a positive control. l qRT-PCR was performed to assess the interaction between FOXC2 and biotin-labeled FOXC2-AS1 after RNA pull-down assay. * P
    Click It Nascent Rna Capture Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/click it nascent rna capture kit/product/Thermo Fisher
    Average 93 stars, based on 1190 article reviews
    Price from $9.99 to $1999.99
    click it nascent rna capture kit - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher click it rna alexa fluor 594 imaging kit
    Ataxin-1 NBs are sensitive to RNase/transcription inhibition in control but not in arsenite stress conditions. Neuro-2a cells were transfected to express GFP-ataxin-1[85Q]. At 24 h post-transfection, cells were treated with arsenite as indicated ( A – C ), followed by treatment with ( A ) Saponin only (0.005%, 15 s) for membrane permeabilization, or Saponin + RNase A (100 μg/ml, 1 h, on ice) for <t>RNA</t> degradation, or ( B ) DMSO only, or Actinomycin D in DMSO (2 μg/ml, 3 h 37 °C). Cells were then fixed and stained with DAPI before CLSM imaging. Zoom images (right panels) correspond to the boxed regions. Representative images are shown from 3 independent experiments. ( C ) RNA synthesis was detected using imaging including the Click-iT RNA <t>Alexa</t> Fluor 594. 5-ethynyl uridine was added for 24 h before arsenite treatment, then incorporated 5-ethynyl uridine was detected (red). Zoom images (right panels) correspond to the boxed regions. ( D ) Quantitative analysis of RNA staining intensity ratio (NB area/non-NB area in the nucleoplasm). Each symbol represents a single data point. Results represent the mean ± SEM (n > 10). Significance values were calculated by unpaired two-tailed student’s t-test, ***p
    Click It Rna Alexa Fluor 594 Imaging Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/click it rna alexa fluor 594 imaging kit/product/Thermo Fisher
    Average 99 stars, based on 221 article reviews
    Price from $9.99 to $1999.99
    click it rna alexa fluor 594 imaging kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    Thermo Fisher click it rna alexa fluor 488 hcs assay
    Protein stability and cell cycle measurements in mouse and human neural progenitors. (A) RT-qPCR analysis of Pax6, Olig2, Nkx2.2 and Isl1 expression in mouse and human differentiations from the Ptchl-mKATE2 targeted stem cell lines. (B) Representative histograms of mKATE2 intensity measurements to estimate protein half-life from mouse (orange) and human (blue) neural progenitors. (C) Representatve histograms of DNA content of mouse and human cells, and dual parameter plots of <t>Alexa</t> Fluor 488-conjugated EdU and DNA indicating the gates to quantify EdU positive cells. (D) Pairwise relationships between proportion of S phase estimations (c 0 ), cell cycle lenght ( T ) and saturation (σ) in mouse and human cells.
    Click It Rna Alexa Fluor 488 Hcs Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/click it rna alexa fluor 488 hcs assay/product/Thermo Fisher
    Average 95 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    click it rna alexa fluor 488 hcs assay - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    91
    Thermo Fisher click it reaction
    Protein stability and cell cycle measurements in mouse and human neural progenitors. (A) RT-qPCR analysis of Pax6, Olig2, Nkx2.2 and Isl1 expression in mouse and human differentiations from the Ptchl-mKATE2 targeted stem cell lines. (B) Representative histograms of mKATE2 intensity measurements to estimate protein half-life from mouse (orange) and human (blue) neural progenitors. (C) Representatve histograms of DNA content of mouse and human cells, and dual parameter plots of <t>Alexa</t> Fluor 488-conjugated EdU and DNA indicating the gates to quantify EdU positive cells. (D) Pairwise relationships between proportion of S phase estimations (c 0 ), cell cycle lenght ( T ) and saturation (σ) in mouse and human cells.
    Click It Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/click it reaction/product/Thermo Fisher
    Average 91 stars, based on 516 article reviews
    Price from $9.99 to $1999.99
    click it reaction - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    84
    Thermo Fisher click it capture nascent rnaclick it nascent rna capture kit
    Protein stability and cell cycle measurements in mouse and human neural progenitors. (A) RT-qPCR analysis of Pax6, Olig2, Nkx2.2 and Isl1 expression in mouse and human differentiations from the Ptchl-mKATE2 targeted stem cell lines. (B) Representative histograms of mKATE2 intensity measurements to estimate protein half-life from mouse (orange) and human (blue) neural progenitors. (C) Representatve histograms of DNA content of mouse and human cells, and dual parameter plots of <t>Alexa</t> Fluor 488-conjugated EdU and DNA indicating the gates to quantify EdU positive cells. (D) Pairwise relationships between proportion of S phase estimations (c 0 ), cell cycle lenght ( T ) and saturation (σ) in mouse and human cells.
    Click It Capture Nascent Rnaclick It Nascent Rna Capture Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/click it capture nascent rnaclick it nascent rna capture kit/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    click it capture nascent rnaclick it nascent rna capture kit - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    Image Search Results


    EVs contain plasmodial RNAs. ( a ) Quantification of P. falciparum RNAs transferred to EVs. Infected RBCs were labeled with Click-It RNA Alexa Fluor 488. After 30 h. of incubation, EVs were collected and EU incorporation was analyzed by Flow Cytometry. ( b ) Distribution of the reads mapped to the P. falciparum genome according to the corresponding chromosomes. ( c ) Distribution of the reads mapped to the P. falciparum according to the size of the chromosome. ( d ) Types of Plasmodium genes in the EVs by RNA-Seq. ( e ) Extracellular vesicles contain a large number of Apicoplast tRNA and Mitochondrial rRNA. ( f ) RNA expression is different in EVs than mixed stage parasite expression pattern. ( g ) EV RNA composition is different that of iRBC. The expression levels shown by RNAseq of EV has no detectable correlation with that of steady state iRBC, showing that EV is not merely a smaller form of iRBC. Neither parametric (Pearson’s R) nor parametric correlations (Kendell’s tau) analysis detected correlation between EV and iRBC at any time points with correlation larger than 0.001. The X-axis shows the average steady state RNA expression levels.

    Journal: Scientific Reports

    Article Title: Malaria infected red blood cells release small regulatory RNAs through extracellular vesicles

    doi: 10.1038/s41598-018-19149-9

    Figure Lengend Snippet: EVs contain plasmodial RNAs. ( a ) Quantification of P. falciparum RNAs transferred to EVs. Infected RBCs were labeled with Click-It RNA Alexa Fluor 488. After 30 h. of incubation, EVs were collected and EU incorporation was analyzed by Flow Cytometry. ( b ) Distribution of the reads mapped to the P. falciparum genome according to the corresponding chromosomes. ( c ) Distribution of the reads mapped to the P. falciparum according to the size of the chromosome. ( d ) Types of Plasmodium genes in the EVs by RNA-Seq. ( e ) Extracellular vesicles contain a large number of Apicoplast tRNA and Mitochondrial rRNA. ( f ) RNA expression is different in EVs than mixed stage parasite expression pattern. ( g ) EV RNA composition is different that of iRBC. The expression levels shown by RNAseq of EV has no detectable correlation with that of steady state iRBC, showing that EV is not merely a smaller form of iRBC. Neither parametric (Pearson’s R) nor parametric correlations (Kendell’s tau) analysis detected correlation between EV and iRBC at any time points with correlation larger than 0.001. The X-axis shows the average steady state RNA expression levels.

    Article Snippet: EU incorporated EV RNA was detected using Click chemistry according to the manufacturer’s protocol (Invitrogen, C10329) and nuclei were counterstained using Hoechst 33342 and analyzed with a MACSQuant VYB flow cytometer.

    Techniques: Infection, Labeling, Incubation, Flow Cytometry, Cytometry, RNA Sequencing Assay, RNA Expression, Expressing

    FOXC2-AS1 positively regulated FOXC2 expression via enhancing FOXC2 mRNA stability. a The relative expression of FOXC2-AS1 in the cytoplasm and nucleus of SW480 and LoVo cells. FISH detection was used to investigate the location of FOXC2-AS1 in cells ( b ) and tissues ( c ). Scale bars = 100 μm. d The expression of FOXC2 was examined in 66 CRC and 15 adjacent normal tissues. e The expression relationship between FOXC2-AS1 and FOXC2 was analyzed in 66 CRC tissues. FOXC2 expression was examined in FOXC2-AS1-silenced SW480 and LoVo cells by qRT-PCR method ( f ) and Western blot ( g ). h qRT-PCR detected the levels of nascent FOXC2 pre-mRNA with Click-iT Nascent RNA Capture Kit in FOXC2-AS1-silenced and control cells. i qRT-PCR investigated FOXC2 mRNA stability in FOXC2-AS1-silenced and control cells treated with the transcriptional inhibitor actinomycin D (50 ng/ml) for different times. j Schematic diagram of FOXC2-AS1 and FOXC2 gene locus and structure. The red grid represents the completely complementary region. The number represents the length of exon or intron. k qRT-PCR was conducted to analyze RNase protection experiment. β-actin was used as a negative control, while PDCD4-AS1 was used as a positive control. l qRT-PCR was performed to assess the interaction between FOXC2 and biotin-labeled FOXC2-AS1 after RNA pull-down assay. * P

    Journal: Cell Death & Disease

    Article Title: LncRNA FOXC2-AS1 enhances FOXC2 mRNA stability to promote colorectal cancer progression via activation of Ca2+-FAK signal pathway

    doi: 10.1038/s41419-020-2633-7

    Figure Lengend Snippet: FOXC2-AS1 positively regulated FOXC2 expression via enhancing FOXC2 mRNA stability. a The relative expression of FOXC2-AS1 in the cytoplasm and nucleus of SW480 and LoVo cells. FISH detection was used to investigate the location of FOXC2-AS1 in cells ( b ) and tissues ( c ). Scale bars = 100 μm. d The expression of FOXC2 was examined in 66 CRC and 15 adjacent normal tissues. e The expression relationship between FOXC2-AS1 and FOXC2 was analyzed in 66 CRC tissues. FOXC2 expression was examined in FOXC2-AS1-silenced SW480 and LoVo cells by qRT-PCR method ( f ) and Western blot ( g ). h qRT-PCR detected the levels of nascent FOXC2 pre-mRNA with Click-iT Nascent RNA Capture Kit in FOXC2-AS1-silenced and control cells. i qRT-PCR investigated FOXC2 mRNA stability in FOXC2-AS1-silenced and control cells treated with the transcriptional inhibitor actinomycin D (50 ng/ml) for different times. j Schematic diagram of FOXC2-AS1 and FOXC2 gene locus and structure. The red grid represents the completely complementary region. The number represents the length of exon or intron. k qRT-PCR was conducted to analyze RNase protection experiment. β-actin was used as a negative control, while PDCD4-AS1 was used as a positive control. l qRT-PCR was performed to assess the interaction between FOXC2 and biotin-labeled FOXC2-AS1 after RNA pull-down assay. * P

    Article Snippet: To investigate whether FOXC2-AS1 regulates FOXC2 transcription, we examined the levels of nascent FOXC2 pre-mRNA with Click-iT Nascent RNA Capture Kit (Life Technologies, USA) in FOXC2-AS1-silenced and control cells.

    Techniques: Expressing, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Western Blot, Negative Control, Positive Control, Labeling, Pull Down Assay

    TFIIIA depletion lead to 5S rRNA neosynthesis inhibition ( A ) Real time qPCR of TFIIIA mRNA 72 hours after the beginning of transfection procedure with specific siRNA against TFIIIA mRNA (siTFIIIA) or non silencing siRNA in HCT116 cells. Graph bars represent the mean ± SEM of three independent experiments. ( B ) Real time qPCR of captured 5-EU labeled 5S rRNA. 72 hours after the beginning of TFIIIA interference, HCT116 cells were labeled with 5-EU for 1 hour and RNA was extracted from siNS, siTFIIIA and from an unlabeled sample, after a chase time of 2 hours with Uridine 1 mM, as indicated above by the representation of the experimental design. All the extracted RNA were modified with biotin azide, captured by streptavidin magnetic beads, reverse-trascribed into cDNA and 5S rRNA relative sequence was amplified by qPCR. Graph bars represents mean ± SEM of three independent experiments. ( C ) Real time qPCR of total 5S rRNA 72 hours after the beginning of transfection procedure with siTFIIIA or siNS, in HCT116 cells. Graph bars represent the mean ± SEM of three independent experiments. ( D ) Polysome profile analysis of whole HCT116 extracts separated by sucrose gradient centrifugation. Samples were collected 72 hours after siNS or siTFIIIA transfection.

    Journal: Oncotarget

    Article Title: The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition

    doi: 10.18632/oncotarget.13833

    Figure Lengend Snippet: TFIIIA depletion lead to 5S rRNA neosynthesis inhibition ( A ) Real time qPCR of TFIIIA mRNA 72 hours after the beginning of transfection procedure with specific siRNA against TFIIIA mRNA (siTFIIIA) or non silencing siRNA in HCT116 cells. Graph bars represent the mean ± SEM of three independent experiments. ( B ) Real time qPCR of captured 5-EU labeled 5S rRNA. 72 hours after the beginning of TFIIIA interference, HCT116 cells were labeled with 5-EU for 1 hour and RNA was extracted from siNS, siTFIIIA and from an unlabeled sample, after a chase time of 2 hours with Uridine 1 mM, as indicated above by the representation of the experimental design. All the extracted RNA were modified with biotin azide, captured by streptavidin magnetic beads, reverse-trascribed into cDNA and 5S rRNA relative sequence was amplified by qPCR. Graph bars represents mean ± SEM of three independent experiments. ( C ) Real time qPCR of total 5S rRNA 72 hours after the beginning of transfection procedure with siTFIIIA or siNS, in HCT116 cells. Graph bars represent the mean ± SEM of three independent experiments. ( D ) Polysome profile analysis of whole HCT116 extracts separated by sucrose gradient centrifugation. Samples were collected 72 hours after siNS or siTFIIIA transfection.

    Article Snippet: Neo-synthesized and pre-existing 5S rRNA evaluation Neo-synthesized and pre-existing RNA amounts were evaluated by Nascent RNA Capture Kit® (Life technologies, Eugene Oregon, USA) following manufacturer's instructions.

    Techniques: Inhibition, Real-time Polymerase Chain Reaction, Transfection, Labeling, Modification, Magnetic Beads, Sequencing, Amplification, Gradient Centrifugation

    Pre-existing 5S rRNA is recruited to MDM2 binding during ribosome biogenesis inhibition ( A ) Graphical experimental design relative to the collection of 5S rRNA fractions from MDM2 immunoprecipitated samples. We labeled HCT116 cells with 5-EU and we performed MDM2 immunoprecipitation on the whole protein extract. The RNA extracted from immunoprecipitated samples was in turn subjected to the capturing of labeled fraction (by Click-it Nascent RNA capture kit, Life technologies, Eugene Oregon, USA) with the collection of the unlabeled fraction or was used directly as a total RNA fraction. RNA fractions were reverse-transcribed and qPCR was performed in order to measure the different amount of 5S rRNA (see also materials and methods). ( B ) Western Blot analysis of MDM2 immuoprecipitation. Whole protein extract was collected from HCT116 cells treated with ACTD (8nM) for 8 hours, 72 hours after siNS or siTFIIIA transfection procedure. Representative Western blots show the level of MDM2 both in INPUT or MDM2 and IGG immunoprecipitated samples (IP). ( C ) Fold changes of neo-synthesized and pre-existing 5S rRNA fractions relative to total 5S rRNA variations bound to MDM2 after TFIIIA interference and ACTD treatment. HCT116 cells were labeled for 8 hours, as indicated above by the experimental design. Whole protein extraction was immunoprecipitated with an antibody against MDM2 and RNA was extracted. Labeled and unlabeled RNA fraction was isolated via click chemistry approach and neo-synthesized, pre-existing and total 5S rRNA amount was evaluated by RT-qPCR (see also experimetal procedures). Graph bars represent mean ± SEM of three independent experiments.

    Journal: Oncotarget

    Article Title: The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition

    doi: 10.18632/oncotarget.13833

    Figure Lengend Snippet: Pre-existing 5S rRNA is recruited to MDM2 binding during ribosome biogenesis inhibition ( A ) Graphical experimental design relative to the collection of 5S rRNA fractions from MDM2 immunoprecipitated samples. We labeled HCT116 cells with 5-EU and we performed MDM2 immunoprecipitation on the whole protein extract. The RNA extracted from immunoprecipitated samples was in turn subjected to the capturing of labeled fraction (by Click-it Nascent RNA capture kit, Life technologies, Eugene Oregon, USA) with the collection of the unlabeled fraction or was used directly as a total RNA fraction. RNA fractions were reverse-transcribed and qPCR was performed in order to measure the different amount of 5S rRNA (see also materials and methods). ( B ) Western Blot analysis of MDM2 immuoprecipitation. Whole protein extract was collected from HCT116 cells treated with ACTD (8nM) for 8 hours, 72 hours after siNS or siTFIIIA transfection procedure. Representative Western blots show the level of MDM2 both in INPUT or MDM2 and IGG immunoprecipitated samples (IP). ( C ) Fold changes of neo-synthesized and pre-existing 5S rRNA fractions relative to total 5S rRNA variations bound to MDM2 after TFIIIA interference and ACTD treatment. HCT116 cells were labeled for 8 hours, as indicated above by the experimental design. Whole protein extraction was immunoprecipitated with an antibody against MDM2 and RNA was extracted. Labeled and unlabeled RNA fraction was isolated via click chemistry approach and neo-synthesized, pre-existing and total 5S rRNA amount was evaluated by RT-qPCR (see also experimetal procedures). Graph bars represent mean ± SEM of three independent experiments.

    Article Snippet: Neo-synthesized and pre-existing 5S rRNA evaluation Neo-synthesized and pre-existing RNA amounts were evaluated by Nascent RNA Capture Kit® (Life technologies, Eugene Oregon, USA) following manufacturer's instructions.

    Techniques: Binding Assay, Inhibition, Immunoprecipitation, Labeling, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Synthesized, Protein Extraction, Isolation, Quantitative RT-PCR

    FoxM1 mRNA is downregulated by Nutlin-3 in TP53 wild type cells but not in TP53 mutant cells Upper panel (A-C) : Cells were treated with vehicle (0.05% DMSO) or 10 μM Nutlin-3 for 3, 6 or 24 h. Lower panel (D-F) : Cells were treated with DMSO, Nutlin-3, CHX, CHX+Nutlin-3, ActD or ActD+Nutlin-3 for 24 h. Total RNA was isolated and subjected to real-time RT-PCR analysis. GAPDH was used for normalization of FoxM1 expression. It is important to note that FoxM1 mRNA is quite stable in the presence of ActD in cell lines with wild type TP53 (D E) . Data are presented as Mean ± SD of 3 experiments. **** indicates P

    Journal: Oncotarget

    Article Title: Targeting of mutant p53-induced FoxM1 with thiostrepton induces cytotoxicity and enhances carboplatin sensitivity in cancer cells

    doi:

    Figure Lengend Snippet: FoxM1 mRNA is downregulated by Nutlin-3 in TP53 wild type cells but not in TP53 mutant cells Upper panel (A-C) : Cells were treated with vehicle (0.05% DMSO) or 10 μM Nutlin-3 for 3, 6 or 24 h. Lower panel (D-F) : Cells were treated with DMSO, Nutlin-3, CHX, CHX+Nutlin-3, ActD or ActD+Nutlin-3 for 24 h. Total RNA was isolated and subjected to real-time RT-PCR analysis. GAPDH was used for normalization of FoxM1 expression. It is important to note that FoxM1 mRNA is quite stable in the presence of ActD in cell lines with wild type TP53 (D E) . Data are presented as Mean ± SD of 3 experiments. **** indicates P

    Article Snippet: Click-iT Nascent RNA Capture and Real-Time RT-PCR analysis A Click-iT Nascent RNA Capture kit (Invitrogen) was used to examine FoxM1 mRNA transcription and stability according to the protocol provided by the manufacturer.

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

    Endogenous wild type p53 suppresses FoxM1 expression whereas R248Q mutant p53 enhances FoxM1 expression (A) Effects of p53 knockdown using shRNAs on FoxM1 protein expression in p53 WT A2780, NCI-H23, and p53 mutant HEC-1A and ES2 cell lines. Endogenous FoxM1 is upregulated when wild type TP53 is knocked down by two shRNAs in A2780 and NCI-H23 cells. Similarly, downregulation of S241F mutant TP53 also upregulate FoxM1 expression, suggesting that S241F mutant retains negative regulatory effect on FoxM1 expression. In contrast, downregulation of R248Q in HEC-1A cells downregulates FoxM1 expression, suggesting that R248Q mutant may have gain of positive regulatory effect of FoxM1 expression. Proteins were isolated from p53 shRNA batch clones and subjected to Western analysis. β-actin was used for normalization of loading. (B) p53 knockdown using shRNAs blocks the downregulation of FoxM1 protein expression by Nutlin-3 in A2780 and NCI-H23 cells. Cells were treated with or without Nultin-3 for 24 h. Proteins were then isolated and subjected to Western analysis. β-actin was used for normalization of loading. (C-D) p53 knockdown blocks the downregulation of FoxM1 mRNA by Nutlin-3 in A2780 and NCI-H23 cells. Cells were treated with or without Nultin-3 for 24 h. Total RNA was isolated and subjected to real-time RT-PCR analysis. GAPDH was used for normalization of FoxM1 expression. Data are presented as Mean ± SD of 3 experiments. * indicates P

    Journal: Oncotarget

    Article Title: Targeting of mutant p53-induced FoxM1 with thiostrepton induces cytotoxicity and enhances carboplatin sensitivity in cancer cells

    doi:

    Figure Lengend Snippet: Endogenous wild type p53 suppresses FoxM1 expression whereas R248Q mutant p53 enhances FoxM1 expression (A) Effects of p53 knockdown using shRNAs on FoxM1 protein expression in p53 WT A2780, NCI-H23, and p53 mutant HEC-1A and ES2 cell lines. Endogenous FoxM1 is upregulated when wild type TP53 is knocked down by two shRNAs in A2780 and NCI-H23 cells. Similarly, downregulation of S241F mutant TP53 also upregulate FoxM1 expression, suggesting that S241F mutant retains negative regulatory effect on FoxM1 expression. In contrast, downregulation of R248Q in HEC-1A cells downregulates FoxM1 expression, suggesting that R248Q mutant may have gain of positive regulatory effect of FoxM1 expression. Proteins were isolated from p53 shRNA batch clones and subjected to Western analysis. β-actin was used for normalization of loading. (B) p53 knockdown using shRNAs blocks the downregulation of FoxM1 protein expression by Nutlin-3 in A2780 and NCI-H23 cells. Cells were treated with or without Nultin-3 for 24 h. Proteins were then isolated and subjected to Western analysis. β-actin was used for normalization of loading. (C-D) p53 knockdown blocks the downregulation of FoxM1 mRNA by Nutlin-3 in A2780 and NCI-H23 cells. Cells were treated with or without Nultin-3 for 24 h. Total RNA was isolated and subjected to real-time RT-PCR analysis. GAPDH was used for normalization of FoxM1 expression. Data are presented as Mean ± SD of 3 experiments. * indicates P

    Article Snippet: Click-iT Nascent RNA Capture and Real-Time RT-PCR analysis A Click-iT Nascent RNA Capture kit (Invitrogen) was used to examine FoxM1 mRNA transcription and stability according to the protocol provided by the manufacturer.

    Techniques: Expressing, Mutagenesis, Isolation, shRNA, Clone Assay, Western Blot, Quantitative RT-PCR

    Ataxin-1 NBs are sensitive to RNase/transcription inhibition in control but not in arsenite stress conditions. Neuro-2a cells were transfected to express GFP-ataxin-1[85Q]. At 24 h post-transfection, cells were treated with arsenite as indicated ( A – C ), followed by treatment with ( A ) Saponin only (0.005%, 15 s) for membrane permeabilization, or Saponin + RNase A (100 μg/ml, 1 h, on ice) for RNA degradation, or ( B ) DMSO only, or Actinomycin D in DMSO (2 μg/ml, 3 h 37 °C). Cells were then fixed and stained with DAPI before CLSM imaging. Zoom images (right panels) correspond to the boxed regions. Representative images are shown from 3 independent experiments. ( C ) RNA synthesis was detected using imaging including the Click-iT RNA Alexa Fluor 594. 5-ethynyl uridine was added for 24 h before arsenite treatment, then incorporated 5-ethynyl uridine was detected (red). Zoom images (right panels) correspond to the boxed regions. ( D ) Quantitative analysis of RNA staining intensity ratio (NB area/non-NB area in the nucleoplasm). Each symbol represents a single data point. Results represent the mean ± SEM (n > 10). Significance values were calculated by unpaired two-tailed student’s t-test, ***p

    Journal: Scientific Reports

    Article Title: Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics

    doi: 10.1038/s41598-020-57994-9

    Figure Lengend Snippet: Ataxin-1 NBs are sensitive to RNase/transcription inhibition in control but not in arsenite stress conditions. Neuro-2a cells were transfected to express GFP-ataxin-1[85Q]. At 24 h post-transfection, cells were treated with arsenite as indicated ( A – C ), followed by treatment with ( A ) Saponin only (0.005%, 15 s) for membrane permeabilization, or Saponin + RNase A (100 μg/ml, 1 h, on ice) for RNA degradation, or ( B ) DMSO only, or Actinomycin D in DMSO (2 μg/ml, 3 h 37 °C). Cells were then fixed and stained with DAPI before CLSM imaging. Zoom images (right panels) correspond to the boxed regions. Representative images are shown from 3 independent experiments. ( C ) RNA synthesis was detected using imaging including the Click-iT RNA Alexa Fluor 594. 5-ethynyl uridine was added for 24 h before arsenite treatment, then incorporated 5-ethynyl uridine was detected (red). Zoom images (right panels) correspond to the boxed regions. ( D ) Quantitative analysis of RNA staining intensity ratio (NB area/non-NB area in the nucleoplasm). Each symbol represents a single data point. Results represent the mean ± SEM (n > 10). Significance values were calculated by unpaired two-tailed student’s t-test, ***p

    Article Snippet: RNA staining For detection of RNA the Click-iT RNA Alexa Fluor 594 imaging kit (ThermoFisher, C10330) was used.

    Techniques: Inhibition, Transfection, Staining, Confocal Laser Scanning Microscopy, Imaging, Two Tailed Test

    Protein stability and cell cycle measurements in mouse and human neural progenitors. (A) RT-qPCR analysis of Pax6, Olig2, Nkx2.2 and Isl1 expression in mouse and human differentiations from the Ptchl-mKATE2 targeted stem cell lines. (B) Representative histograms of mKATE2 intensity measurements to estimate protein half-life from mouse (orange) and human (blue) neural progenitors. (C) Representatve histograms of DNA content of mouse and human cells, and dual parameter plots of Alexa Fluor 488-conjugated EdU and DNA indicating the gates to quantify EdU positive cells. (D) Pairwise relationships between proportion of S phase estimations (c 0 ), cell cycle lenght ( T ) and saturation (σ) in mouse and human cells.

    Journal: bioRxiv

    Article Title: Species-specific developmental timing is associated with global differences in protein stability in mouse and human

    doi: 10.1101/2019.12.29.889543

    Figure Lengend Snippet: Protein stability and cell cycle measurements in mouse and human neural progenitors. (A) RT-qPCR analysis of Pax6, Olig2, Nkx2.2 and Isl1 expression in mouse and human differentiations from the Ptchl-mKATE2 targeted stem cell lines. (B) Representative histograms of mKATE2 intensity measurements to estimate protein half-life from mouse (orange) and human (blue) neural progenitors. (C) Representatve histograms of DNA content of mouse and human cells, and dual parameter plots of Alexa Fluor 488-conjugated EdU and DNA indicating the gates to quantify EdU positive cells. (D) Pairwise relationships between proportion of S phase estimations (c 0 ), cell cycle lenght ( T ) and saturation (σ) in mouse and human cells.

    Article Snippet: EU-incorporated RNAs were labelled using Click-iT™ RNA Alexa Fluor 488 HCS Assay (Thermo Fisher Scientific C10327), and AHA-incorporated proteins were labeled using Click-iT™ Cell Reaction Buffer Kit (Thermo Fisher Scientific C10269) on a volume of 150ul per sample reaction.

    Techniques: Quantitative RT-PCR, Expressing