Journal: Bioscience Reports

Article Title: The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation

doi: 10.1042/BSR20150073

Figure Lengend Snippet: Differentiation-induced changes in Tfam mRNA stability ( A ) The nt sequence corresponding to the 3′-UTR of mouse Tfam is illustrated. Possible binding sites for RNA-binding proteins, HuR, CuGBP1 and KSRP are underlined. Primers used for 746 and 600 bp Tfam 3′-UTR constructs are highlighted in grey for the forward reaction and black for reverse reaction. ( B ) Linear regression analysis of Tfam mRNA availability in actinomycin D-treated day 1 compared with day 4 differentiated C 2 C 12 cells ( n =3). Slopes of day 1 (−14.45±2.657) and day 4 (−4.475±1.039) decay rates differentiated cells are 3.2-fold different ( n =3 experiments). ( C ) Effect of differentiation on Renilla luciferase activities of 746 and 600 bp Tfam 3′-UTRs. (Day 3–4 compared with day 0–2; n =4, ¶ P

Article Snippet: Second, fully differentiated C2 C12 myotubes were treated with either 10 μg/ml actinomycin D to inhibit transcription or methanol as a vehicle-matched control, for 2, 4 or 6 h. Total RNA was extracted with TRIZOL reagent (Invitrogen) according to manufacturer's instructions.

Techniques: Sequencing, Binding Assay, RNA Binding Assay, Construct, Luciferase

Journal: Journal of Virology

Article Title: Role of Herpes Simplex Virus ICP27 in the Degradation of mRNA by Virion Host Shutoff RNase ▿

doi: 10.1128/JVI.00975-10

Figure Lengend Snippet: Shutoff of protein synthesis in HEp-2, Vero, and HeLa cells infected with the ΔICP27 mutant virus. Replicate 25-cm 2 cultures of HEp-2 (A), Vero (B), or HeLa (C) cells were either mock infected (lane 1) or infected with 20 PFU per cell of HSV-1(F) (lane 2) or the ΔU L 41 (lane 3), ΔICP27 (lane 4), or ΔICP4 (lane 5) mutant virus at 37°C in the presence of 10 μg actinomycin D per ml. After 1 h, the inoculum was replaced with fresh medium containing actinomycin D. Two hours after removal of the inoculum, the cells were labeled for 30 min with [ 35 S]methionine before being harvested. Whole-cell lysates were resolved on a 12% denaturing polyacrylamide gel, dried, and subjected to autoradiography. The intensities of the bands were quantified with the aid of a General Dynamics Storm phosphorimager and are reported as percentages (shown) of the band intensities of mock-infected cells.

Article Snippet: Total RNA was extracted at the times indicated in Fig. after actinomycin D addition with the aid of TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Infection, Mutagenesis, Labeling, Autoradiography

Journal: Journal of Virology

Article Title: Role of Herpes Simplex Virus ICP27 in the Degradation of mRNA by Virion Host Shutoff RNase ▿

doi: 10.1128/JVI.00975-10

Figure Lengend Snippet: Analysis of cellular mRNA decay at an early time (3 h) after infection. (A) Schematic representation of the experiment. (B) Replicate 25-cm 2 cultures of HeLa cells were either mock infected (lanes 1 to 4) or infected with 10 PFU per cell of HSV-1(F) (lanes 5 to 8) or the ΔU L 41 (lanes 9 to 12) or ΔICP27 (lanes 13 to 16) mutant virus for 1 h. The inoculum was then replaced with fresh complete medium, and the cell cultures were incubated at 37°C for two additional hours. Transcription was then stopped by addition of 10 μg of actinomycin D (Act. D) per ml (time zero). The cells were harvested at the time intervals indicated, and total mRNA was extracted. The accumulation of the GAPDH and IEX-1 transcripts was visualized by Northern blotting. The ethidium bromide staining of 18S rRNA is shown as a loading control. Letters on the right indicate IEX-1 bands A, B, and C. (C) The intensities of the bands at various time points were quantified by phosphorimager analysis in three independent experiments, and the mRNA remaining is plotted as a percentage of that in mock-infected cells (GAPDH mRNA) or relative to time after addition of actinomycin D (IEX-1 mRNA) (averages ± standard errors).

Article Snippet: Total RNA was extracted at the times indicated in Fig. after actinomycin D addition with the aid of TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Techniques: Infection, Mutagenesis, Incubation, Activated Clotting Time Assay, Northern Blot, Staining

Journal: The Journal of Cell Biology

Article Title: The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins

doi: 10.1083/jcb.200403069

Figure Lengend Snippet: Inhibition of S6K restores IRS-1 mRNA. (A) IRS-1 mRNA levels (top) in serum-starved TSC2 +/+ or TSC2 −/− MEFs in untreated cells or after the addition of 20 nM rapamycin for various times. The blot was reprobed for β-actin as a control for mRNA loading (bottom). White line indicates that intervening lanes have been removed. (B) IRS-1 mRNA levels (top) in TSC2 −/− MEFs treated with 20 nM rapamycin alone, or in the presence of 10 μg/ml actinomycin D for the final 10 h of a 24-h treatment. The blot was reprobed for β-actin as a control for mRNA loading (bottom). (C) Quantitative RT-PCR analysis of untreated, rapamycin-treated, and rapamycin plus actinomycin D–treated TSC2 −/− MEFs. Left, β-actin; right, IRS-1. A single graph from triplicate determinations showing identical results is shown. (D) RNAi-mediated inhibition of S6K1 and S6K2. Western blotting of extracts from TSC2 +/+ or TSC2 −/− MEFs transfected with a combination of scrambled siRNAs (C) or S6K1, S6K2, or S6K1 plus S6K2 siRNAs. Top, S6K1; middle, S6K2; bottom, anti-pS6 (Ser240/244). Where indicated, cells were stimulated for 10 min with insulin or treated with 20 nM rapamycin for 1 h. (E) IRS-1 mRNA levels in TSC2 +/+ MEFs or TSC2 −/− MEFs transfected with a combination of S6K1 and S6K2 scrambled siRNAs (C), S6K1, or S6K2 siRNAs, or treated for 24 h with 20 nM rapamycin. Top, IRS-1 mRNA; The blot was also reprobed for β-actin as a control for mRNA loading (bottom). (F) Quantitative RT-PCR analysis of untreated TSC2 +/+ , TSC2 −/− , or TSC2 −/− treated with 20 nM rapamycin or RNAi to S6K1 or S6K2. Left, β-actin; right, IRS-1. A single graph from triplicate determinations showing identical results is shown.

Article Snippet: For the actinomycin D experiment, TSC2 −/− cells were starved for 24 h in the absence or presence of 20 nM rapamycin or for 24 h with rapamycin with addition of 10 μg/ml actinomycin D for the last 10 h. Quantitative PCR was performed using the JOE-labeled mouse β-actin LUX™ Primer set (Invitrogen) and an IRS-1–specific primer pair with a 6-FAM–labeled probe synthesized by QIAGEN.

Techniques: Inhibition, Quantitative RT-PCR, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: CIRBP is a novel oncogene in human bladder cancer inducing expression of HIF-1α

doi: 10.1038/s41419-018-1109-5

Figure Lengend Snippet: CIRBP increases mRNA stability and protein translation of HIF-1α in BCa cells. a qRT-PCR analysis for the effects on the HIF-1A mRNA levels of CIRBP knockdown and CIRBP overexpression. b Western blot analysis of HIF-1α performed on CIRBP knockdown and CIRBP overexpression UM-UC-3 cells, exposed (+) or not (−) to CoCl 2 (100 μM for 4 h, Sigma 15862). c Immunofluorescence staining revealed alterations of HIF-1α (green) after 48 h transfection of siCIRBP in UM-UC-3 cells or CIRBP overexpression plasmid in UM-UC-3 cells under normoxia (CIRBP red). Nuclei were stained by DAPI (blue). d 24 h after transfection with siCIRBP (pretreated with CoCl 2 ) or CIRBP overexpression plasmid (under normoxia), UM-UC-3 cells were cultured in the presence of Actinomycin D (15 μg/ml, Abcam ab141058), qRT-PCR detected mRNA levels of HIF-1A specific time points of 0, 2, 4, 6 h. GAPDH is used as the normalization control. e RNA-Binding Protein Immunoprecipitation (RIP) assay was performed with anti-CIRBP or anti-IgG, qRT-PCR detected the IP efficiency (percent input). f Biotin pull-down assay was achieved to confirm the interacts of CIRBP and HIF-1A mRNAs 3′-UTR, both 3′-UTR and 5′-UTR of HIF-1A mRNA transcripts were constructed with biotin-label, transcripts of GAPDH mRNA were used as a negative control. Means ± standard deviation from three independent experiments. T -test was used for statistical analysis

Article Snippet: In order to examine the effects of CIRBP on the stability of the mRNAs of HIF-1A, 24 h after transfection, Cells were pretreated with CoCl2 (100 μM, Sigma c8661) for 4 h, then Actinomycin D (15 μg/ml, Abcam ab141058) was added to inhibit transcription.

Techniques: BIA-KA, Quantitative RT-PCR, Over Expression, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Cell Culture, RNA Binding Assay, Immunoprecipitation, Pull Down Assay, Construct, Negative Control, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Long noncoding RNA EMS connects c-Myc to cell cycle control and tumorigenesis

doi: 10.1073/pnas.1903432116

Figure Lengend Snippet: EMS connects c-Myc to cell cycle control and tumorigenesis. ( A ) A549 cells were infected with lentiviruses expressing control shRNA (shctrl), c-Myc shRNA (shc-Myc), or both c-Myc shRNA and EMS. Forty-eight hours later, cells were incubated with actinomycin

Article Snippet: The following reagents and antibodies were used in this study: DAPI (Sigma), Lipofectamine 2000 (Invitrogen), complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor mixture (Roche Applied Science), actinomycin D (ab141058; Abcam), propidium iodide (ST511; Beyotime), streptavidin-coated agarose beads (Thermo Fisher Scientific), antibodies against actin (sc-47778,1:1,000; Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sc-166545, 1:5,000; Santa Cruz Biotechnology), Flag (F3165, 1:4,000; Sigma), E2F1 (3742S, 1:1,000; Cell Signaling), c-Myc for ChIP assay (9402S; Cell Signaling), c-Myc for Western blotting (9402S, 1:1,000; Cell Signaling), RALY (A302-070A, 1:1,000; Bethyl), RARP (sc-8007, 1:1,000; Santa Cruz Biotechnology), and horseradish peroxidase-conjugated secondary antibodies against rabbit (111-035-144, 1:10,000; Jackson Immunoresearach) and mouse (115-035-062, 1:10,000; Jackson ImmunoResearch).

Techniques: Infection, Expressing, shRNA, Incubation

Journal: Nucleic Acids Research

Article Title: MPK-1/ERK pathway regulates DNA damage response during development through DAF-16/FOXO

doi: 10.1093/nar/gky404

Figure Lengend Snippet: mpk-1 mutation suppress csb-1 mutant sensitivity to transcription blocking lesions. ( A ) L1 larvae were treated 48 h with different concentrations of illudinM, in liquid culture, then plated and larval stages were determined immediately. ( B ) L1 larvae were treated 24 h with different concentrations of actinomycin D, in liquid culture, then plated and larval stages were determined 24 h later at 25°C. ( C ) L1 larvae were treated 24 h with different concentrations of α-amanitin, in liquid culture, then plated and larval stages were determined 24 h later at 25°C. Error bars represent standard deviation. * P

Article Snippet: Actinomycin D and α-amanitin sensitivity assay Synchronized L1 larvae were obtained by hypochlorite treatment and were treated with indicated concentrations of actinomycin D (SIGMA-ALDRICH A1410) or α-amanitin (SIGMA-ALDRICH A2263) in M9 medium with OP50 for 24 h at 20°C on a shaking platform.

Techniques: Mutagenesis, Blocking Assay, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Protein fishing from single live cells

doi: 10.1186/s12951-018-0395-5

Figure Lengend Snippet: Protein fishing from HeLa cells. Sampling of HeLa cells 24 h after actinomycin D treatment. a Cytoplasmic or b nuclear extract was deposited on grid-marked, APTES-coated coverslips, followed by protein detection using a cocktail of anti-β-actin-FITC and anti-p53-APC. The coverslips were imaged under the FITC and APC filter channels, sequentially

Article Snippet: Actinomycin D treatment Actinomycin D (2 mg/mL) stock from Sigma-Aldrich was diluted in 90% DMEM, with 10% FBS to a final concentration of 50 nM.

Techniques: Sampling

Journal: Gene Therapy

Article Title: A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer

doi: 10.1038/gt.2014.61

Figure Lengend Snippet: MHC II antigenic presentation by DCs to specific CD4+ T cells. C57BL/6 mice and Sgca-/- mice were injected i.m. with 5 × 10 9  vg of rAAV1_CMV_SGCA-HY (gray histogram) or rAAV1_CMV_SGCA-HY-Mir142T (black histogram) vectors into both TA. After 5 or 8 days, popliteal and inguineal lymph node were harvested from five mice per group to select CD11c+ cells by magnetic cell sorting and culture them with CFSE-stained Dby-specific CD4+ T cells for 3 days at 37 °C. Cell division was analyzed by FACS in live CD4+ T cells after sequentially gating on lymphoid cells in the culture (upper left scatter plot), and then on live CD4+ 7-actinomycin D (7AAD)-negative cells (upper right scatter plot). Lower panel histograms show the levels of CFSE in CD4+ T cells in one representative experiment out of three.

Article Snippet: Dead cells were excluded using 7-actinomycin D (Sigma Chemical Co., St Louis, MO, USA) staining.

Techniques: Mouse Assay, Injection, FACS, Staining

Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: ANTIVIRAL EFFECTS OF SAIKOSAPONINS ON HUMAN CORONAVIRUS 229E IN VITRO

doi: 10.1111/j.1440-1681.2006.04415.x

Figure Lengend Snippet: Efects of saikosaponin A (), B 2 (▪), C (▨) and D (▥) and actinomycin D (□) on the viability of the human fetal lung fibroblast (MRC‐5) cell line. Cells were treated with various concentrations (0, 2.5, 25 and 250 µmol/L) of saikosaponins A, B 2 , C and D for 96 h. Cell viability was determined by the XTT assay. Data are the mean±SD of three independent experiments. * P

Article Snippet: Chemicals Actinomycin D, dimethylsulphoxide (DMSO) and saikosaponins A, B2 , C and D ( ) were purchased from Sigma Chemical (St Louis, MO, USA).

Techniques: XTT Assay

Journal: Journal of Biomedical Science

Article Title: Nontranscriptional activation of PI3K/Akt signaling mediates hypotensive effect following activation of estrogen receptor ? in the rostral ventrolateral medulla of rats

doi: 10.1186/1423-0127-19-76

Figure Lengend Snippet: Effects of RNA synthesis inhibitor on the ERβ agonist-induced vasodepressive responses. Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, or to rats pretreated with actinomycin D (AMD, 5 or 10 nmol) or 5 % DMSO, administered into the bilateral RVLM 1 hour before DPN (2 pmol) microinjection. Values are presented as the mean ± SEM; n = 7-8 animals per experimental group. * P

Article Snippet: The test agents used in this study included 17β-estradiol-3-sulphate sodium (E2β; Sigma–Aldrich, St. Louis, MO, USA); a selective ERα agonist, 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1 H-pyrazole (PPT; Tocris Cookson Inc., Bristol, UK); a selective ERβ agonist, diarylpropionitrile (DPN; Tocris Cookson); a nonspecific ER antagonist, ICI 182780 (Tocris Cookson); a selective ERα antagonist, methyl-piperidino-pyrazole (MPP; Tocris Cookson); a selective ERβ antagonist, R,R-tetrahydrochrysene (R,R-THC; Tocris Cookson); a transcription inhibitor, actinomycin D (AMD; Tocris Cookson); a PI3K inhibitor, LY294002 (Calbiochem, San Diego, CA, USA); an Akt inhibitor (Calbiochem); an antisense oligonucleotide (ASON) against the rat ERα or ERβ mRNA (Quality Systems, Taipei, Taiwan) or the scrambled (SCR) ERα or ERβ oligonucleotide (Quality Systems).

Techniques:

Journal: Cellular Signalling

Article Title: Iron depletion suppresses mTORC1-directed signalling in intestinal Caco-2 cells via induction of REDD1

doi: 10.1016/j.cellsig.2016.01.014

Figure Lengend Snippet: Effects of modulating REDD1 expression on DFO-induced changes in mTORC1 signalling in Caco-2 cells. (A) Caco-2 cells were incubated in the absence or presence of 100 μM DFO for 16 h prior to incubation with 100 μM Actinomycin D, a transcriptional inhibitor for time periods indicated. At the end of these treatments cells were lysed and lysates immunblotted with antibodies against HIF-1α, REDD1, p70S6K Thr 389 , p70S6K and actin. REDD1 and p70S6K Thr 389 abundance was quantified using ImageJ and data presented as mean ± SEM from at least three experiments. The asterisks signify significant differences (p 

Article Snippet: The transcription inhibitor actinomycin D was purchased from Tocris (Bristol, UK) and the proteasome inhibitor MG132 was from Selleck Chemicals (Houston, USA).

Techniques: Expressing, Incubation

Journal: Stem Cell Research & Therapy

Article Title: Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

doi: 10.1186/s13287-019-1532-2

Figure Lengend Snippet: Effect of cLIUS on select genes, SOX9 localization, and SOX9 mRNA stability. Gene expression of a c-MYC and c-JUN and b SOX9 and RUNX2 in MSCs exposed to cLIUS at indicated frequencies were estimated by qRT-PCR ( n = 3). MSCs plated at 1 × 10 5 cells/well were exposed to cLIUS (14 kPa) at frequencies: 5 MHz (2.5 Vpp), 2 MHz (6 Vpp), or 8 MHz (9.5 Vpp) at constant pressure amplitude of 14 kPa one time for 5 min. Non-cLIUS-stimulated MSCs served as controls ( n = 3). Data are shown as the mean ± standard deviation (Welch’s t -test). c MSCs in coverslips ( n = 3) at an initial seeding density of 1 × 10 5 cells/well were stimulated with cLIUS application at 14 kPa (5 MHz, 2.5 Vpp) for 5 min and fixed in 4% PFA. Confocal micrographs (× 60 magnification) of immunofluorescent staining of SOX9 (green) shows the localization of SOX9 in the MSCs under cLIUS (Scale bar = 20 μm). Nucleus was stained by Dapi (blue) and d quantified by ImageJ ( n = 10–20). Data are shown as the mean ± standard deviation. e MSCs (2 × 10 4 cells per well) were treated with 5 μg/ml actinomycin D, followed by stimulation with cLIUS at 14 kPa (5 MHz, 2.5 Vpp), for 15 min ( n = 3). Total RNA was collected after 0 min, 30 min, 1 h, and 2 h of actinomycin D treatment, and the fold change in mRNA transcripts of SOX9 were quantified by qRT-PCR. In a parallel experiment without actinomycin D treatment, total RNA was collected at indicated time points following cLIUS stimulation, and the fold change in SOX9 mRNA transcripts was quantified by qRT-PCR ( n = 3). Non-cLIUS-stimulated samples served as controls at respective time points ( n = 3). Data represent the average ± standard deviation of fold change in SOX9 mRNA levels normalized to time point 0. f Graphical representation of the amount of SOX9 mRNA transcripts in actinomycin D-treated MSCs with or without cLIUS stimulation normalized to non-actinomycin D-treated samples at respective time points

Article Snippet: After cLIUS stimulation, the medium was immediately removed and replaced with serum-free DMEM containing 5 μg/ml actinomycin D (Cell Signaling Technology, USA).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Staining