Nematode growth medium agar plates containing inhibitors were prepared by mixing molten NGM agar with 0.3 μg/ml cycloheximide (Sigma, Saint Louis, MO, USA), 0.3 μg/ml anisomycin (A.G. Scientific, San Diego, CA, USA), or 0.1 μg/ml actinomycin D (MP Biomedicals, Solon, OH, USA) (final concentration). One day before experiments, plates were spread with OP50, and were left at RT overnight. Animals were cultivated on the plates for 2 h at 20°C before spaced training, or for 4 h at 20°C before massed training. Under the conditions, ∼50% of protein synthesis of the animal was inhibited by this treatment (Amano and Maruyama, 2011 (link)).
Actinomycin d
Manufactured by MP Biomedicals
Sourced in United States
Actinomycin D is a naturally occurring antibiotic compound produced by the bacterium Streptomyces parvulus. It is a potent inhibitor of RNA synthesis, specifically by binding to DNA and preventing the initiation of RNA transcription.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (4 mentions)
Rnaseout, by Thermo Fisher Scientific (4 mentions)
Iscript gdna clear cdna synthesis kit, by Bio-Rad (2 mentions)
Actinomycin d, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Fugene hd, by Promega (2 mentions)
Direct zol rna miniprep plus kit, by Zymo Research (2 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ampure rna clean beads, by Beckman Coulter (1 mentions)
User enzyme, by New England Biolabs (1 mentions)
Truseq rna kit, by Illumina (1 mentions)
Ampure xp bead purification, by Beckman Coulter (1 mentions)
Truseq rna sample preparation kit v2, by Illumina (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Control sirna, by Thermo Fisher Scientific (1 mentions)
Sihur, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna protocol, by Illumina (1 mentions)
13 protocols using actinomycin d
1
Nematode Growth Inhibitor Assay
2
Transcriptional Inhibition by Actinomycin D
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Cells were seeded in a six‐well plate. The actinomycin D (SKU:02104658‐CF, MP Biomedicals, CA, USA), an mRNA transcriptional inhibitor, was added into the wells, and cells were collected at 0 h, 2 h, 4 h, and 6 h after treatment. Total RNA was extracted from cells using an RNeasy Kit (QIAGEN). The purified mRNA was subjected to qPCR analysis. The house‐keep gene HPRT1 was used as the internal control.
3
RNA-seq Library Preparation Protocol
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2ug total RNA was used for cDNA library preparation by using a modified protocol based on the Illumina Truseq RNA Sample Preparation Kit V2. After poly-A selection, fragmentation, and priming, reverse transcription was carried out for 1st strand cDNA synthesis in the presence of RNaseOut (Invitrogen) and actinomycin-D (MP Biomedicals). The synthesized cDNA was further purified by using AMPure RNAClean beads (Beckman Coulter). A modified method by incorporation of dUTP instead of dTTP was prepared and used for the second strand synthesis. After AMPure XP bead purification (Beckman Coulter), following the standard protocol recommended by the Illumina Truseq RNA kit, end repairing, A-tailing, and ligation of index adaptors were sequentially performed for generation of cDNA libraries. After size selection of libraries using Pippin Prep (SAGE Biosciences), the dUTP-containing strands were destroyed by digestion with USER enzymes (New England Biolabs) followed by PCR enrichment. Final cDNA libraries were analyzed in Agilent Bioanalyzer and quantified by Quant-iT Pico-Green assays (Life Technologies) before sequencing using the HiSeq platform (Illumina).
4
Measuring mRNA Stability in PC3 Cells
5
Glutathione Redox Regulation Assay
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L-buthionine-S,R-sulfoximine (BSO), Glutathione (GSH), oxidized Glutathione (GSSG) and DL-Dithiothreitol (DTT) were purchased from Sigma-Aldrich. 35S-Translabel (1000 C/mmol), containing labeled methionine and cysteine, and Actinomycin D were products of MP Biochemicals. Guanidine HCl, GSH Sepharose 4B, and Sepharose 4B, were purchased from Qiagen, BioWorld, and GE, respectively.
6
Regulation of Chemokine Signaling in LX-2 Cells
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LX-2 cells were transduced with either adeno-NOX4 or control virus (adeno-CMV) (Applied Biological Materials Inc. Richmond, BC). To knock down HuR, LX-2 cells were transfected with either siHuR or control siRNA (Thermo Fisher, Waltham, MA) using RiboJuice transfection reagent (MilliporeSigma) following the instruction. The cells then were treated with 2.0 μg/ml actinomycin D (MP Biomedicals, Solon, OH) at the indicated time points. Total RNA was extracted with the TRIzol reagent (Life Technologies) following the manufacturer’s instructions. The amount of CCR2 and CCL2 mRNA was measured by RT-qPCR. mRNA half-life was calculated as T1/2 = Ln(2)/λ.
7
RNA-seq Library Preparation with Illumina TruSeq
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RNA was isolated from cells using the RNeasy kit (Qiagen). Libraries were generated using 1μg of total RNA using a modified Illumina TruSeq Stranded mRNA protocol. Reverse transcription was performed with the addition of RNaseOut (Invitrogen) and actinomycin-D (MP Biomedicals). The resulting product was cleaned using AMPure RNAClean beads. Additionally, second-strand synthesis was performed using dUTP instead of dTTP and an additional USER (New England Biolabs) digestion step was incorporated after size-selection. Libraries were sequenced at 101x6x0x101 on a HiSeq (Illumina) to a minimum depth of 30 million reads per sample.
8
Nos2 3' UTR-B Reporter Assay
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IEC6 cells were seeded on 60 mm dishes. The following day, control and Zfp36-specific shRNAs were induced in rIEC6 cells with 1 μg/mL doxycycline (Acros Organics). After 24 hours cells were transfected with 4 μg of the pmirGLO Nos2 3′ UTR-B reporter plasmid and 4 μg pBluescript. The next day, cells were treated with 5 μg/mL Actinomycin D (MP Biomedicals) for 0, 4, or 8 hours. Subsequently, total RNA was isolated and converted to cDNA, as described above, and firefly luciferase transcript levels were measured by quantitative PCR using the indicated primers (Table S2 ). The data is presented as percent mRNA remaining with renilla luciferase expression serving to normalize values.
9
Measuring mRNA Stability in PC3 Cells
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PC3 cells were plated at 35,000 cells per well in 24-well plates and transfected 24 h later using 250ng pLuc2CP-noARE, 2ng pRL-UBC, and 2μL Fugene HD (Promega). Each construct was transfected into two or three replicate wells per time point and the entire experiment was repeated 3 times. After 24 h of transfection, cells were treated with 5μg/μL Actinomycin D (MP Biomedicals), then collected in 250μL TRIzol (Invitrogen) at 0, 8, and 24 h after Actinomycin D treatment. Samples were immediately processed upon collection using the Direct-zol RNA Miniprep Plus Kit (Zymo Research) including on-column DNase treatment. cDNA synthesis was performed on equal volumes of RNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad), introducing a second round of DNase treatment with this kit. qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) with primers amplifying firefly luciferase mRNA (Table S7 , primers #30–31).
10
Quantifying Transcript Stability via Actinomycin D
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Actinomycin D (MP Biomedicals) at a final concentration of 100 μg/ml was added to the mid-exponential culture to inhibit transcription. Cells were then collected after 0, 5, 10, 15 and 30 min post-treatment, and total RNA was extracted. For mono- and poly-cistronic operons, Northern blot and primer extension assays, respectively, were used to determine RNA stability as described above. The quantity of transcript at each time point was calculated based on band intensity using the Quantity One software (Bio-Rad). RNA decay rate was determined by the residual RNA at a given time point over that at time 0. RNA half-life was calculated based on the exponential regression curve as described (35 (link)), where the abundance of the initial transcript decreased to 50%.
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