Whole mount Drosophila ovary samples (5-10 ovaries per sample) were either treated with 0.2-2 μg/ml of Actinomycin D diluted in Grace’s medium, 250 μg/ml Alpha-amanitin (Sigma-Aldrich, A2263) in Grace’s medium or the same dilution of DMSO for 10 min prior to, or were directly incubated with 2 mM 5-ethynyl uridine (EU) for 15 min and washed twice with Grace’s medium. Samples were fixed in 4% paraformaldehyde diluted in Grace’s medium for 15 min. Samples were washed 6 times for 10 min each with Triton Wash (1X PBS, 0.5% Triton X-100), and then rinsed once with 1X PBS before incubating for 30 min in the Click-iT reaction cocktail, prepared according to product specifications (Thermo Fisher Scientific, Click-iT™ RNA Alexa Fluor™ 488 Imaging Kit, C10329). Samples were rinsed twice with Click-iT Reaction Rinse Buffer and twice with 1X PBS, and then washed for 30 min in 1X PBS. Samples were either stained for antibodies starting at the antibody wash step (see above) or immediately stained with Hoechst 33342 (1:1000 in PBS) for 15 min to label DNA and then rinsed 3 times in 1X PBS before mounting as described in the immunofluorescence section.
Alpha amanitin
Manufactured by Merck Group
Sourced in Canada
Alpha-amanitin is a laboratory product developed by Merck Group. It is a cyclic peptide compound derived from the death cap mushroom, Amanita phalloides. The core function of alpha-amanitin is to serve as a research tool for studying the inhibition of RNA polymerase II, an essential enzyme involved in the transcription of genetic information.
Automatically generated - may contain errors
Lab products found in correlation
Actinomycin d, by Merck Group (3 mentions)
Ruthenium red, by Merck Group (2 mentions)
Azd6244, by Cayman Chemical (2 mentions)
Bapta am, by Merck Group (2 mentions)
U0126, by Merck Group (2 mentions)
A23187, by Merck Group (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
Click it rna alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Selleck Chemicals (1 mentions)
Melphalan hydrochloride, by Merck Group (1 mentions)
5 flurouracil 5 fu, by Merck Group (1 mentions)
Radioactively labeled nucleoside triphosphates ntps, by PerkinElmer (1 mentions)
Puromycin, by Merck Group (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Ampliscribe t7 flash transcription kit, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Liberase dl, by Merck Group (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
14 protocols using alpha amanitin
1
Labeling Drosophila Ovary RNA Synthesis
2
Anticancer Drug Screening in Cell Lines
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Cell culture medium was replaced with medium containing indicated concentrations of drugs for indicated time periods. All drugs were dissolved in DMSO, and DMSO in the absence of inhibitor was used as a control in all experiments. Substances in the initial experiments were from the “approved oncology drugs set IV” library obtained from the National Cancer Institute (NCI), USA (https://wiki.nci.nih.gov/display/NCIDTPdata/Compound+Sets ). The after substances were purchased: Melphalan hydrochloride (Y0001158, Sigma), Etiposide (S1225, Selleckchem), Cisplatin (S1166, Selleckchem), actinomycin D (A9415, Sigma), alpha‐amanitin (A2263, Sigma) and PARP1 inhibitor A‐966492 (S2197, Selleckchem).
3
Synthesis and Assays of NI Compounds
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All NI were synthesized by Gilead Sciences, Inc. Puromycin, 5-flurouracil (5-FU), and alpha-amanitin were purchased from Sigma-Aldrich (St. Louis, MO). All radioactively labeled nucleoside triphosphates (NTPs) were purchased from PerkinElmer (Shelton, CT).
4
Pharmacological Modulation of Calcium Signaling
5
Pharmacological Modulation of Calcium Signaling
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The following drugs were used in experiments: 10 μM gadolinium III chloride (Gd3+) (for both zebrafish and cell culture) to block Piezo1, and 30μM Ruthenium Red or 10μM BAPTA-AM were used to inhibit calcium (all from Sigma). 10 μM Roscovitine was used to block CDK1. 5μM alpha-amanitin (Sigma) was used to inhibit RNA polymerase II and III. 1μM AZD6244 (Cayman Chemical) or 1μM U0126 (Sigma) were used to inhibit MEK1 and 2. 1μM A23187 or 5μM Thapsigargin (both from Sigma) were used to induce calcium influx.
6
Generation and Analysis of pre-miR-144 Variants
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To generate different pre-mir-144 hairpins universal oligo containing T7 promoter was annealed with specific oligo encoding indicated mutations in the mir-144 stem loop and then filled by PCR. PCR products were purified using Monarch PCR&DNA Cleanup Kit (NEB) and used directly in in vitro transcription reaction performed overnight with AmpliScribe T7-Flash Transcription kit (Epicentre). pre-mir-144 variants were purified from denaturing urea polyacrylamide gel. LNA-modified pre-mir-144 hairpins were ordered from IDT. Pre-miRNA hairpins were injected into single-cell stage zebrafish embryos (1 nl of 10 μM stock) alone or together with 1 nL of 0.2 mg/ml alpha-amanitin (Sigma-Aldrich). Total RNA was extracted at 6 h after injection from 20 embryos using Trizol (Invitrogen) and their processing was analyzed by Northern blotting.
7
Metastatic Tumor Dissection and Isolation
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Lungs were perfused with PBS through the right ventricle, and metastases (<1 mm in diameter) were dissected under a dissection microscope and pooled from 1–5 mice. The tissues were subsequently digested using an enzyme mix of Liberase DL (Sigma-Aldrich 5466202001, 0.52 U/mL), TL (Sigma-Aldrich 5401020001, 0.26 U/mL) and DNase I (Sigma-Aldrich DN25, 150 µg/mL) for 30 min at 37°C. For sorting for RNA-seq or qRT-PCR, transcription inhibitors alpha-amanitin (Sigma-Aldrich A2263, 5 µg/mL) and actinomycin D (Sigma-Aldrich A1410, 1 µg/mL) were also added to the digestion buffer. Flow cytometry was analyzed using a LSRII cytometer (BD Biosciences), and the data were analyzed using Flowjo software (TreeStar, v10; a possible free alternative is Flowing Software 2.5.1 from University of Turku). FACSAria II (BD Biosciences) and Moflo Astrios (Beckman Coulter) were used for sorting. Antibody information is found in
Table 1 .
8
Optimizing Inhibitor Concentrations in Cell Studies
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All inhibitors were resuspended in DMSO to recommended effective concentrations (Bensaude, 2011 (link)). Flavopiridol hydrochloride hydrate (F3055, Sigma‐Aldrich) was resuspended to a stock concentration of 12.5 mM and diluted 1:12,500 to an effective concentration of 1 μM. Actinomycin D (A1510, Sigma‐Aldrich) was resuspended to an initial concentration of 1 mg/ml and diluted 1:200 to an effective concentration of 5 μg/ml. Triptolide (T3652, Sigma‐Aldrich) was resuspended to a stock concentration of 10 mM and diluted 1:20,000 to an effective concentration of 500 nM. The effectiveness of all inhibitors was verified on the basis of Pol II phosphorylation changes at the whole nucleus level (Appendix Fig S14 ). Alpha‐amanitin (A2263, Sigma‐Aldrich) was micro‐injected into the yolk (1 nl per embryo) at a concentration of 0.2 mg/ml (dissolved in water) at the 1‐cell stage (Joseph et al, 2017 (link); Hilbert et al, 2021 (link)). Whole embryo flavopiridol treatment was carried out by adding 10 μM flavopiridol to the embryo media (Vopalensky et al, 2018 (link)).
9
Ovary Dissection and Alpha-Amanitin Incubation
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Whole ovaries dissected from flies fatten with yeast paste were incubated in Grace’s medium containing 50 µg/mL of alpha-amanitin (Sigma) for the indicated time points before RNA was extracted.
10
Alpha-Amanitin Treatment and Gene Expression
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A total of 100 000 cells were plated in a six‐well and treated with 50 μg·mL−1 of alpha‐amanitin (Sigma, Oakville, ON, Canada, A2263), as described (Khalil et al., 2008 ). Total RNA was extracted at time‐points 0, 2, 5, 6, and 10 h postincubation, and gene expression was analyzed by qPCR as previously described.
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