GM06990 lymphoblastoid cells were mainained in RPMI 1640 (Life Technologies; 11875093) supplemented with 15% fetal bovine serum (Thermo Scientific; SH40007-13), 2mM L -Glutamine (Life Technologies; 25030-081) and Pen Strep (Life Technologies; 15140-122) at 37 C in 5% CO2. To avoid the onset of aneuploidy, cultures were generally exchanged for a fresh thaw every 4-6 weeks. For the actinomycin D treatment, cells were incubated for 2 hrs with 200 ng/ml actinomycin D (BioVision; 1036-50) and then harvested, washed in PBS and deposited on polylysine coated slides for fixation.
Actinomycin d
Manufactured by Abcam
Sourced in United States, United Kingdom
Actinomycin D is a polypeptide antibiotic that binds to DNA and inhibits RNA synthesis. It is used as a research tool in cell and molecular biology laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Rnase r, by Solarbio (2 mentions)
Rnase r, by Illumina (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Rnase r, by Abcam (1 mentions)
Mg132, by Merck Group (1 mentions)
Enzalutamide mdv3100, by Selleck Chemicals (1 mentions)
Normal human astrocytes, by BNCC (1 mentions)
Dulbecco modified eagle medium (dmem), by Procell (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Ab176916, by Abcam (1 mentions)
Streptavidin coated agarose beads, by Thermo Fisher Scientific (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
M2 beads, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
37 protocols using actinomycin d
1
Actinomycin D Treatment of GM06990 Cells
2
Investigating mRNA Stability with YTHDF2
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The experiments were performed as previously described with slight modification.30 (link) To examine the effects of YTHDF2 expression on the stability of the mRNAs of interest, actinomycin D (5 μg/mL, Abcam, ab141058) was added to inhibit transcription after 24 h transfection. Total RNA was harvested at specific time points of 0 h, 2 h, and 4 h, then RT-PCR detected mRNA levels.
3
Profiling circLDLR Stability in Hepatocytes
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The circLDLR stability was detected by performing RNase R and Actinomycin D assays. Total RNA (10 μg) of lysed Hepa1-6 cells was treated with 40 U RNase R (Solarbio, Beijing, China) at 37 °C for 15 min. RNase-free total RNA was utilized to be a control. The circLDLR and LDLR levels were then determined using qRT-PCR.
Hepa1-6 cells (5 × 104 cells/well) were seeded on 6-well plates and treated with Actinomycin D (Abcam, Cambridge, MA, USA) for 0, 8, 16 and 24 h. After that, the circLDLR and LDLR expression was detected via qRT-PCR.
Hepa1-6 cells (5 × 104 cells/well) were seeded on 6-well plates and treated with Actinomycin D (Abcam, Cambridge, MA, USA) for 0, 8, 16 and 24 h. After that, the circLDLR and LDLR expression was detected via qRT-PCR.
4
Investigating CircATAD2 Stability in Breast Cancer
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MCF-7 or MDA-MB-231 cells were treated with 1 μg/ml actinomycin D (Abcam) at indicated times [0, 3, and 6 h]. Then, total RNA of the BC cells was extracted. To identify circular characteristics of circATAD2 stability, the total RNA (2 μg) was incubated with or without RNase R (6 U, 3 U/μg) (Epicentre, SanDiego, CA, USA) at 37 °C for 30 min. The relative expression level of circATAD2, ATAD2 were determined using qRT-PCR.
5
Actinomycin D-Induced RNA Dynamics
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TE‐1 cells were placed in five wells in 24‐well plates (5 × 104 cells per well). Actinomycin D was applied to cells after 24 h (2 μg/ml, Abcam, Cambridge, UK) for 0, 4, 8, 12, and 24 h, respectively. After that, the relative RNA levels of circIMMP2L and mIMMP2L were analyzed by qRT‐PCR and normalized to the values of the 0 h group.
6
Evaluating circFAM13B, FAM13B, and PKM2 Expression
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T24 or UMUC3 cells were treated with 1 μg/ml actinomycin D (Abcam, UK) at indicated times (0, 2, 4, 6, 8, and 10 h). Then, the total RNA of these cells was extracted, and the expression levels of circFAM13B, FAM13B, and PKM2 were determined using qRT-PCR.
7
Measuring Circular RNA and mRNA
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After Y-79 cells (5 × 104 cells/well) were seeded into 24-well plates and incubated overnight, 2 μg/mL actinomycin D (Abcam) was added to block transcription at indicated time points (0, 4, 8, 12 and 24 h). Next, qRT-PCR was conducted to examine the expression of hsa_circ_0007534 and DDX42 mRNA.
8
Circularization and Transcription Inhibition
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About 2 μg of total RNAs was incubated with 3 U/μg of RNase R or not for 30 min at 37°C. The resulting RNAs were collected and analyzed by qRT‐PCR.
To block transcription, HPASMCs were treated with 2 μg/mL actinomycin D (Abcam, Cambridge, MA, USA) in 24‐well plates, and then, qRT‐PCR was adopted to test circST6GAL1 and ST6GAL1 mRNA expression levels.
To block transcription, HPASMCs were treated with 2 μg/mL actinomycin D (Abcam, Cambridge, MA, USA) in 24‐well plates, and then, qRT‐PCR was adopted to test circST6GAL1 and ST6GAL1 mRNA expression levels.
9
Analyzing circBCAR3 and BCAR3 Expression
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Two micrograms of total RNA was cultured with 5 U/μg RNase R (#ab286929, Abcam) for half an hour at 37 °C. EC109 and KYSE150 cells were treated with 2 μg/mL actinomycin D (#ab291108, Abcam) for 1, 2, 4, 8, 12 h. RNA expression of circBCAR3 and linear BCAR3 was analyzed using qRT-PCR.
10
Actinomycin D Effects on circAKT3
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The Actinomycin D assay was performed as previously described [16 (link)]. SGC7901CDDP and BGC823CDDP cells were seeded in 5 wells in 24-well plates (5 × 104 cells per well). Twenty-four hours later, the cells were exposed to Actinomycin D (2 μg/ml, Abcam, ab141058) for 0 h, 6 h, 12 h, 18 h and 24 h. The cells were then harvested, and the relative RNA levels of circAKT3 and AKT3 mRNA were analyzed by RT-qPCR and normalized to the values measured in the group in the 0 h group (mock treatment).
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