Journal: Molecular cell

Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses

doi: 10.1016/j.molcel.2016.12.025

Figure Lengend Snippet: BDNF Post-translationally Induces Lin28a, but Not Paralog Lin28b, through Protein Stabilization (A) Quantification (top) and representative immunoblot (bottom) of Lin28a protein levels in lysates from hippocampal neurons exposed to BDNF with or without transcription blockade (Actinomycin-D) for the indicated times, normalized to GAPDH and plotted relative to 0-min BDNF without Actino-mycin-D (set as 1.0). #p

Article Snippet: On DIV 14–19, hippocampal cultures were changed into Serum Reduced Media (NBA + 0.5% B27 + glutamine) for 2 hr prior to stimulation, followed by pre-incubation with Actinomycin-D (Life Technologies A7592) at a final concentration of 0.5 μg/ml for 10 min if required.

Techniques:

Journal: Nature biotechnology

Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

doi: 10.1038/s41587-019-0090-6

Figure Lengend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

Article Snippet: For experiments testing the stability of circRNAs, 5 μg/mL actinomycin D (Sigma Aldrich A9415) was added to cells for 6 h prior to extraction of RNA.

Techniques: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

Journal: Nature biotechnology

Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

doi: 10.1038/s41587-019-0090-6

Figure Lengend Snippet: Improved inhibition of NF-κB pathway by circRNA aptamers. a , Design of circular RNAs that contain NF-κB pathway-inhibiting aptamers. Circular RNAs are designed to contain a F30 3-way junction (black) with Broccoli on one arm, and an NF-κB aptamer on the other arm, while the circularizing stem forms at the base of the F30 3-way junction. This design allows for functional investigation of pathway-inhibiting aptamers while also probing for abundance of this circular RNA using Broccoli fluorescence. b , Tornado efficiently expresses the NF-κB aptamer as a racRNA in HEK293 cells. HEK293 cells were transfected with a plasmid expressing the NF-κB aptamer as a linear RNA or a a racRNA for two days. Cells were then treated with actinomycin D (ActD) for 6 h and RNA was harvested to detect aptamer expression levels before and after actinomycin D treatment. Aptamer levels were detected based on in-gel staining using DFHBI-1T to detect the Broccoli incorporated into the RNA. The Tornado-expressed racRNA generates a single bright Broccoli-fluorescent bands that is resistant to actinomycin D treatment, while the linear band is barely detected (outlined in white and shown in a brightness-adjusted image on the right). c , The NF-κB aptamer is an effective pathway inhibitor when expressed using the Tornado expression system. IL-1β-induced NF-κB pathway activation was detected by luminescence in cells encoding luciferase driven by a NF-κB-promoter. Cells expressed either the circular or linear NF-κB aptamer as indicated. IL-1β-induced luminescence is 8% inhibited by linear expression and 70% inhibited by the circular expression. Circular RNA-expressing cells that were pre-sorted for aptamer expression based on their green fluorescence shows more efficient pathway inhibition (~85%). Luminescence was normalized according to the number of cells present during the assay. All signals were normalized to that of the cells expressing RNA without the NF-κB aptamer after activation. Data in this panel represent the mean (n = 3 or 4 stimulation and assay independent samples).

Article Snippet: For experiments testing the stability of circRNAs, 5 μg/mL actinomycin D (Sigma Aldrich A9415) was added to cells for 6 h prior to extraction of RNA.

Techniques: Inhibition, Functional Assay, Fluorescence, Transfection, Plasmid Preparation, Expressing, Staining, Activation Assay, Luciferase

Journal: Nature

Article Title: SCFFbw7 Regulates Cellular Apoptosis By Targeting Mcl-1 for Ubiquitination and Destruction

doi: 10.1038/nature09732

Figure Lengend Snippet: Elevated Mcl-1 expression protects Fbw7-deficient T-ALL cell lines from ABT-737-induced apoptosis a , Cell viability assays showing that Fbw7-deficient T-ALL cell lines were more sensitive to sorafenib, but resistant to ABT-737 treatment. T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib or ABT-737 for 48 hours before performing the cell viability assays. Data was shown as mean ± SD for three independent experiments. b , Immunoblot analysis of the indicated human T-ALL cell lines with or without ABT-737 (0.8 µM) treatment. c , Specific depletion of endogenous Mcl-1 expression restored ABT-737 sensitivity in the indicated Fbw7-deficient T-ALL cell lines. Various T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of ABT-737 for 48 hours before performing the cell viability assays, or with or without ABT-737 (0.8 µM) treatment for 24 hours before collecting whole cell lysates for immunoblot analysis with the indicated antibodies. For cell viability assays, data was shown as mean ± SD for three independent experiments. d , 7-Amino-Actinomycin D (7-AAD)/Annexin V double-staining FACS analysis to detect the percentage of ABT-737-induced apoptosis in the indicated Fbw7-deficient T-ALL cell lines where the endogenous Mcl-1 was depleted by lentiviral shRNA treatment (lentiviral shGFP was used as a negative control). Various T-ALL cells were cultured in 10% FBS-containing medium with or without ABT-737 (0.8 µM) treatment for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells. e , 7-AAD/Annexin V double-staining FACS analysis to demonstrate that sorafenib treatment restores ABT-737 sensitivity to Fbw7-deficient HPB-ALL cells. HPB-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib and/or ABT-737 for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells.

Article Snippet: For detection of apoptosis, cells treated with various drugs were stained with propidium iodide (Roche), or co-stained with Annexin V-PE and 7-amino-actinomycin D (Annexin V-PE Apoptosis Detection Kit I, BD Bioscience) according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Double Staining, FACS, shRNA, Negative Control