Actinomycin d

Manufactured by Fujifilm

Sourced in Japan, Germany

Actinomycin D is a laboratory-grade chemical compound used in research applications. It functions as an antibiotic and antitumor agent, inhibiting DNA-dependent RNA synthesis.

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28 protocols using actinomycin d

Cytokine-induced IL-6 mRNA expression

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HeLa or ZC3-KO cells were stimulated with 10 pg/mL IL-1ß for 30 min followed by a treatment with 10 µg/mL actinomycin D (Wako) for 2 h. Samples were collected at 0, 30 min, and 2 h post-actinomycin D addition. Total RNA was isolated from the collected cells using the TRI reagent (Sigma). IL-6 mRNA expression was analyzed using northern blot and RT-qPCR.

Darweesh M., Younis S., Hajikhezri Z., Ali A., Jin C., Punga T., Gupta S., Essand M., Andersson L, & Akusjärvi G. (2022). ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression. Frontiers in Immunology, 13, 1002823.

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Antioxidant and Stress Response Compounds

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Carnosic acid, carnosol, and rosmarinic acid were supplied by Nagase Co LTD. (Kobe, Japan). Piciferic acid was purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethylsulfoxide, actinomycin D, cycloheximide, tunicamycin, and hemin were obtained from Wako Pure Chemicals (Osaka, Japan). The tert-butylhydroquione (tBHQ) was obtained from Kanto Chemical (Tokyo, Japan). ISRIB was obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against ATF4 (CREB2) (C-20), Nrf2 (H-300), lamin B (M-20), and HSP90α/β (AC88) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and primary antibodies against eIF2α (9722S) and phospho-Ser51 eIF2α (9721S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies against rabbit and goat immunoglobulin G (IgG) were obtained from Life Technologies (Carlsbad, CA, USA) and Santa Cruz Biotechnology, respectively. hemin was purchased from ICN Biomedicals (Irvine, CA, USA). hemin stock solution (100×) was made by dissolving hemin in 20 mM NaOH.

Mimura J., Inose-Maruyama A., Taniuchi S., Kosaka K., Yoshida H., Yamazaki H., Kasai S., Harada N., Kaufman R.J., Oyadomari S, & Itoh K. (2019). Concomitant Nrf2- and ATF4-Activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes. International Journal of Molecular Sciences, 20(7), 1706.

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MCF-7 Cell Growth Factor and Inhibitor Assay

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MCF-7 cells were maintained in DMEM medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% FBS. Prior to growth hormone treatment, the cells were serum-starved for 16–24 h, and then EGF (PeproTech House, London, UK) or HRG-β 176-246 was added for further analysis. After incubation with the growth factors for the indicated time, the cells were washed twice with PBS. In the case of inhibitor assays, cells are treated with 500 nm of U0126 (Calbiochem, San Diego, CA, USA) or 5 μg·mL⁻¹ of actinomycin D (Wako Pure Chemical Industries, Tokyo, Japan) for the indicated times. Cells were then lysed with Bio-Plex lysis buffer (Bio-Rad Laboratories, Hercules, CA, USA) for western blot analysis. Cells were lysed with RNA lysis buffer (Nucleo Spin RNA II; Macherey-Nagel GmBH & Co., Düren, Germany) for quantitative real time-PCR (qRT-PCR).

Nagashima T., Inoue N., Yumoto N., Saeki Y., Magi S., Volinsky N., Sorkin A., Kholodenko B.N, & Okada-Hatakeyama M. (2015). Feedforward regulation of mRNA stability by prolonged extracellular signal-regulated kinase activity. The Febs Journal, 282(4), 613-629.

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Cell Culture and Conditional Knockout Assays

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DT40 cells were cultured at 38.5 °C under 5% CO₂ in DMEM (Wako) supplemented with 10% fetal bovine serum, 1% chicken serum, penicillin-streptomycin (Gibco) and 10 µM 2-mercaptoethanol (Wako). HeLa cells were maintained as described previously [19 (link)]. For glucose deprivation, DT40 cell pellet was washed twice with 10 mL of phosphate-buffered saline PBS (−) and cultured in DMEM medium without glucose (Gibco), supplemented as described above. For the selective inhibition of the Pol I activity, HeLa cells were treated with actinomycin D (Wako) at 5 nM for 3 h. Dead cells were counted by Trypan blue staining. For RNAi-mediated ARP6 knockdown, HeLa cells were transfected with siRNAs using RNAiMAX (Invitrogen). The cells were analyzed 48 h after transfection. The siRNA duplexes used are: HSS148894 for siARP6 (i) and HSS148895 for siARP6 (ii) (Invitrogen). The target sequence for the control (siControl) is: CUUACGCUGAGUACUUCGA (GL3, Nippon EGT). To repress the expression of the tetracycline-responsive transgenes in DT40 cells, ARP6 and H2A.Z conditional knockout cell lines (ARP6-KO and H2A.Z-KO) were treated with tetracycline (Wako) at a final concentration of 2 µg/mL [11 (link)]. The cells were analyzed 4–6 days (ARP6-KO) and 3–4 days (H2A.Z-KO) after the tetracycline treatment, when the proteins were almost absent [11 (link)].

Kitamura H., Matsumori H., Kalendova A., Hozak P., Goldberg I.G., Nakao M., Saitoh N, & Harata M. (2015). The actin family protein ARP6 contributes to the structure and the function of the nucleolus. Biochemical and biophysical research communications, 464(2), 554-560.

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Measurement of Residual RNA Levels

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LTED cells were treated with 1 μg/mL actinomycin D (Wako) for 15 min to stop RNA synthesis, and then the residual RNA levels were measured at every 60 min for 300 min. RNA isolation, RT, and PCR analysis were performed as described above. cMyc was used as a control for unstable RNA. The primer sets are listed in Supplementary Table 4.

Abdalla M.O., Yamamoto T., Maehara K., Nogami J., Ohkawa Y., Miura H., Poonperm R., Hiratani I., Nakayama H., Nakao M, & Saitoh N. (2019). The Eleanor ncRNAs activate the topological domain of the ESR1 locus to balance against apoptosis. Nature Communications, 10, 3778.

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Quantifying E-selectin Expression in HUVECs

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HUVECs were cultured as described above and treated with 1 ng/ml TNFα for 2 h, followed by actinomycin D (5 µg/ml) (Wako Pure Chemical Industries) for 0, 0.5, and 2 h. RNA was then isolated using an RNeasy Mini Kit (Qiagen) and transcribed into cDNA using the PrimeScript RT Master Mix (Takara Bio). Quantitative PCR analysis was performed using a KAPA SYBR FAST Universal qPCR Kit (Kapa Biosystems) and the Thermal Cycler Dice (Takara Bio). We used the following primer pairs: E-selectin forward, 5′-AAGCCCACATGTGAAGCTGT-3′, and reverse, 5′-CTCCAATAGGGGAATGAGCA-3′; GAPDH forward, 5′-AGTGGATATTGTTGCCATCAAT-3′, and reverse, 5′-CTTGACGGTGCCATGGAATT-3′. The relative expression levels of E-selectin were normalized against those of GAPDH.

Hamada K., Osaka M, & Yoshida M. (2014). Cell Density Impacts Epigenetic Regulation of Cytokine-Induced E-Selectin Gene Expression in Vascular Endothelium. PLoS ONE, 9(4), e90502.

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Collagen and TGF-β1 Regulation

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Antibodies against type I collagen (1:1000, Cat.#1310-01) and β-actin (1:1000, Cat.#sc-47778) were purchased from Southern Biotechnologies (Birmingham, AL, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. Recombinant human TGF-β1 (2 ng/mL, Cat.#240-B-002) was obtained from R&D Systems (Minneapolis, MN, USA). Actinomycin D (Cat.#018-21264) was purchased from Wako (Osaka, Japan).

Sawamura S., Makino K., Ide M., Shimada S., Kajihara I., Makino T., Jinnin M, & Fukushima S. (2022). Elevated Alpha 1(I) to Alpha 2(I) Collagen Ratio in Dermal Fibroblasts Possibly Contributes to Fibrosis in Systemic Sclerosis. International Journal of Molecular Sciences, 23(12), 6811.

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RNA Synthesis Inhibition Dynamics

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Cells of HepG2, PLC/PRF/5, and HeLa cell lines were cultured in DMEM medium supplemented with penicillin at 100 mg/L, streptomycin at 100,000 U/L and 10% fetal bovine serum (Biological Industries Ltd., Israel) in a humidified atmosphere containing 5% CO₂ at 37°C. The cells for culture were dispersed in 60 mm dishes with 7.5 × 10⁶ cells in 5 mL of the medium at overnight and were collected for DNA and total RNA extraction. After washing with phosphate buffered saline, the confluent cells were treated in 0.25% w/v trypsin and 1 mM EDTA at 37°C for 5 min, followed by adjustment to 1 × 10⁵ cells in 1 mL of DMEM medium.
To arrest RNA synthesis, the medium was replaced with fresh medium containing 5 μM actinomycin-D (Wako Pure Chemical Industries Ltd., Osaka, Japan). Cells were harvested at 0, 2.5, 5, 7.5, and 10 h after addition of the transcriptional inhibitor. Then they were examined using real-time PCR.

Inaoka Y., Osawa M., Mukasa N., Miyashita K., Satoh F, & Kakimoto Y. (2015). Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism. Disease Markers, 2015, 865053.

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Manipulating Chromatin Structure and DNA Replication

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For inhibiting DNA replication, 50–100 µM APH (Wako; 011-09811) and 5–15 µM p27 recombinant protein were simultaneously added to the extracts with demembranated sperm chromatin. Geminin recombinant protein (0.4–1.2 µM) was added into the CSF extract and preincubated for 15 min at 22°C, in advance of adding CaCl₂, cycloheximide, and demembranated sperm chromatin. Both recombinant proteins were kindly gifted by Atsuya Nishiyama (University of Tokyo). For manipulating the chromatin structure, 5−20 mM MgCl₂, 10 µg/ml actinomycin D (Wako; 018-21264), or 141 µM ICRF-193 (Santa Cruz; sc-200889) was added to the extract containing preassembled nuclei after 30–40 min of incubation. For digesting DNA inside the nucleus, 0.2 U/µl EcoRI, 0.2 U/µl XhoI, or 0.1 U/µl HaeIII (NEB; R3101, R3101, R0146) was added with the equipped buffer (NEB; 1× CutSmart buffer) into the extract containing preassembled nuclei.

Heijo H., Shimogama S., Nakano S., Miyata A., Iwao Y, & Hara Y. (2020). DNA content contributes to nuclear size control in Xenopus laevis. Molecular Biology of the Cell, 31(24), 2703-2717.

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Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) Cell Line

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The human Müller glial cell line Moorfields/Institute of Ophthalmology‐Müller 1 (MIO‐M1) was provided from Dr G. Astrid Limb (UCL Institute of Ophthalmology, London, United Kingdom).14 The cells were cultured in DMEM containing 10% fetal bovine serum (Thermo Fisher Scientific). For hypoxic exposure, cells were cultured in a gas mixture composed of 1% O₂, 5% CO₂ and 94% N₂. Aldosterone and streptozotocin were from Sigma‐Aldrich. RU486 and MG132 were from Cayman Chemical. Dexamethasone sodium phosphate, triamcinolone acetonide and actinomycin D were from FUJIFILM Wako Pure Chemical Corporation.
Specific siRNAs against TSC22D3 (hs.Ri.TSC22D3.13.1), DUSP1 (hs.Ri.DUSP1.13.3) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L.11 Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.

Kanda A., Hirose I., Noda K., Murata M, & Ishida S. (2020). Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization. Journal of Cellular and Molecular Medicine, 24(8), 4589-4599.

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