r0461 br utp biotium  (Biotium)

Journal: Nucleic Acids Research

Article Title: A circular RNA-gawky-chromatin regulatory axis modulates stress-induced transcription

doi: 10.1093/nar/gkae157

Figure Lengend Snippet: MRE circRNAs are potent inhibitors of stress-induced transcription. ( A ) Schematic overview of the strategy for constructing MRE circRNA stable cell lines. Briefly, the DNA sequences for producing circCG32369(2 ) and circMCPH1(2,3,4,5 ) were individually inserted between the Nhe I and Xma I sites of the Hy_pAct5C laccase2 MCS exon vector, a plasmid modified from ( , ). The generated overexpression vectors, harboring a hygromycin resistance (HygroR) cassette, were then used for stable cell selection with the assistance of hygromycin B for at least 3 weeks. ( B ) RT-qPCR quantification of circCG32369(2 ) and circMCPH1(2,3,4,5 ) verifying their overexpression efficiencies in each stable cell line. The regular S2 cell control was set to ‘1’. Data were generated from six independent experiments and are shown as means ± SEM. Each replicate is represented as a black dot. ( C ) RT-qPCR quantification of MT mRNAs in copper-treated regular S2 and MRE circRNA stable cells. The regular S2 cell control was set to ‘1’. Data were generated from three independent experiments and are shown as means ± SEM. Each replicate is represented as a black dot. P value was calculated by Student's t -test (** P < 0.01; * P < 0.05). (D, E) RNA FISH assays of MtnA and MtnB mRNAs in copper-treated regular S2 and MRE circRNA stable cells ( D ). Representative images from three independent confocal imaging analyses are shown. Scale bar = 5 μm. The relative expression levels of MtnA and MtnB mRNAs were then quantified from 50 cells and are shown in the violin plots ( E ). Lines inside each violin plot represent the first quartile, median and third quartile of the data. P value was calculated by Student's t -test (** P < 0.01; * P < 0.05). ( F ) RT-qPCR quantification of four control circRNAs ( circeIF5B(7,8 ) , circrl(6,7,8 ) , circPde11(3,4,5 ) and circCG17715(3,4,5,6,7) ) and two MRE linear RNAs (linear CG32369 and MCPH1 ) verifying their overexpression efficiencies in the indicated stable cell lines. The regular S2 cell control was set to ‘1’. Data were generated from three independent experiments and are shown as means ± SEM. Each replicate is represented as a black dot. ( G ) RT-qPCR quantification of MT mRNAs in copper-treated regular S2 and stable cells described in (F). The regular S2 cell control was set to ‘1’. Data were generated from three independent experiments and are shown as means ± SEM. Each replicate is represented as a black dot. P value was calculated by Student's t -test (** P < 0.01; * P < 0.05). ( H ) Schematic representation of the NRO assay, adapted from , CC BY 4.0 ( https://creativecommons.org/licenses/by/4.0/ ). ( I ) NRO-qPCR analyses of the newly-synthesized (BrUTP-labeled) MT mRNAs (i.e. evaluation of the transcription activities of the MT genes) in copper-treated regular S2 and MRE circRNA stable cells. The reference genes 18S , Act42A and αTub84B were used as negative controls to assess the technological artifact in each replicate. Data were generated from three independent experiments and are shown as means ± SEM. Each replicate is represented as a black dot. P value was calculated by Student's t -test (** P < 0.01; * P < 0.05).

Article Snippet: In short, after subcellular fractionation, nascent transcripts in nuclei were labeled with bromouridine triphosphate (BrUTP, Sigma, B7166) for 5 min in NRO Buffer (50 mM Tris–HCl pH 7.5, 5 mM MgCl 2 , 150 mM KCl, 0.1% (w/v) srkosyl, 10 mM dithiothreitol and 80 units/ml RNase inhibitor (Beyotime, R0102)), isolated by anti-BrUTP (Abcam, ab1893) following the standard immunoprecipitation procedure and subjected to RT-qPCR assays.

Techniques: Stable Transfection, Plasmid Preparation, Modification, Generated, Over Expression, Selection, Quantitative RT-PCR, Imaging, Expressing, Synthesized, Labeling