Ponasterone a

Manufactured by Thermo Fisher Scientific

Sourced in United States

Ponasterone A is a naturally occurring steroidal compound that can be used as a research tool in cell biology and biochemistry laboratories. It functions as an ecdysteroid agonist, capable of binding and activating ecdysone receptors in various biological systems.

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13 protocols using ponasterone a

Inducing TP53INP1α Expression in U2OS Cells

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In TP53INP1α-inducible U2OS cells, TP53INP1α-GFP expression was induced on 70% confluent cells using 10 μM ponasterone A (Life Technologies) for 24 h. DNA transfections were performed using FuGENE HD (Promega) according to the manufacturer's instructions on 75% confluent cells 24 h before experiment.

Seillier M., Pouyet L., N'Guessan P., Nollet M., Capo F., Guillaumond F., Peyta L., Dumas J.F., Varrault A., Bertrand G., Bonnafous S., Tran A., Meur G., Marchetti P., Ravier M.A., Dalle S., Gual P., Muller D., Rutter G.A., Servais S., Iovanna J.L, & Carrier A. (2015). Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1. EMBO Molecular Medicine, 7(6), 802-818.

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Inducible Fhit expression in H1299 cells

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The engineered H1299 lung carcinoma cells, which inducibly express Fhit cDNA and protein (D1) or an empty vector (E1) have been described [20 (link)]. H1299 cells were maintained in DMEM medium with 10% FBS, zeocin, gentamicin and geneticin. Fhit expression was induced by addition of ponasterone A (2 μM) (Life Technologies) to the growth medium for 48 h.

Kiss D.L., Baez W., Huebner K., Bundschuh R, & Schoenberg D.R. (2017). Impact of FHIT loss on the translation of cancer-associated mRNAs. Molecular Cancer, 16, 179.

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Quantifying Anti-AAV8 Neutralizing Antibodies

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For determination of anti-AAV8 NAb titer in serum, an in vitro reporter system was used as previously described.^{33 (link)} In brief, 96-well plates were seeded with 2 × 10⁴ 2V6.11 cells/well and incubated in DMEM with 10% fetal calf serum at 37°C and 5% CO₂ for 24 h in the presence of ponasterone A (Life Technologies, Carlsbad, CA). Recombinant AAV8-CMV-luciferase (AAV8-CMV-Luc) was diluted in serum-free DMEM (Life Technologies) and incubated with threefold serial dilutions (1:1–1:3,160) of the serum samples, and then incubated for 1 h at 37°C. Subsequently, the serum–vector mixtures were added to the 2V6.11 cells, and after 24 h, cells were lysed with the Bright Glo system (Promega, Madison, WI) and the luciferase activity was measured on a luminometer (ENSPIRE™; Perkin Elmer, Waltham, MA). The NAb titer was reported as the highest serum dilution that inhibited AAV transduction by ≥50% compared with the control without serum (100% transduction). Anti-AAV8 NAb titer was assessed in two independent experiments for all subjects. Any sample with a detectable anti-AAV8 NAb titer (≥1:1) in both experiments was determined as seropositive. Also NAb cross-reactivity to AAV5 and AAV2 was assessed using this in vitro reporter method using AAV5-CMV-Luc and AAV2-CMV-Luc in samples that were positive for anti-AAV8 NAbs.

Aronson S.J., Veron P., Collaud F., Hubert A., Delahais V., Honnet G., de Knegt R.J., Junge N., Baumann U., Di Giorgio A., D'Antiga L., Ginocchio V.M., Brunetti-Pierri N., Labrune P., Beuers U., Bosma P.J, & Mingozzi F. (2019). Prevalence and Relevance of Pre-Existing Anti-Adeno-Associated Virus Immunity in the Context of Gene Therapy for Crigler–Najjar Syndrome. Human Gene Therapy, 30(10), 1297-1305.

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Quantitative HIV Entry via Inducible Affinofile Cells

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CD4 and CCR5 dual-expressing 293-T Affinofile cells were induced and infected in a matrix layout as previously described [90 (link), 91 (link)]. Briefly, 1.2x10⁵ or 1x10⁴ cells were seeded per well of a 24-well culture plate for quantitative flow cytometry of CD4 and CCR5 expression levels, or 96-well clear-bottom culture plate for infections, respectively, and allowed to adhere overnight before being treated with perpendicular serial dilutions of Doxycycline (Sigma Aldrich, Missouri, USA) and Ponasterone A (Invitrogen, Massachusetts, USA) to induce CD4 and CCR5 expression, respectively. Surface expression levels of CD4 and CCR5 were quantified between 18-24h post induction by quantitative flow cytometry using QuantiBRITE beads and PE-labelled antibodies specific for CD4 (clone Q4120; Sigma Aldrich, Missouri, USA) and CCR5 (clone 2D7; BD Biosciences, New Jersey, USA). Affinofile cells were infected 18-24hrs post-induction using 10⁵ RLU of virus per well (calculated by titration on TZM-bl cells) in the presence of 10 μg/ml Diethylaminoethyl (DEAE; Ammersham plc, UK) and luciferase readout was performed after 48hrs of infection.

Beauparlant D., Rusert P., Magnus C., Kadelka C., Weber J., Uhr T., Zagordi O., Oberle C., Duenas-Decamp M.J., Clapham P.R., Metzner K.J., Günthard H.F, & Trkola A. (2017). Delineating CD4 dependency of HIV-1: Adaptation to infect low level CD4 expressing target cells widens cellular tropism but severely impacts on envelope functionality. PLoS Pathogens, 13(3), e1006255.

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Polyglutamine Aggregation Assay in PC12 Cells

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Experiments were performed in inducible PC12 cells (14A2.5) expressing truncated huntingtin exon 1 containing 103Q repeats and fused to EGFP (36 (link)). Cells were maintained in DMEM, 10% horse serum, 5% fetal bovine serum, 1% penicillin/streptomycin with continual selection in 200 μg/ml geneticin. For the aggregation assay, cells were induced with 5 μm ponasterone A (Invitrogen, Carlsbad, CA, USA) for 30 h prior to treatment with compounds. Stock solutions of actinomycin D (Sigma A1410), tunicamycin (Sigma) and YM1 were prepared in DMSO. Cycloheximide (Sigma) was prepared in ddH₂O at 1 mg/ml immediately prior to use. Compounds were added to culture media at the final concentrations indicated. Aggregates were assessed 48 h after polyQ induction by fluorescence microscopy on a Nikon Ti-E microscope equipped with a Coolsnap HQ2 CCD camera (Photometrics, Tuscon, AZ, USA). Aggregates were quantified with the spot detection feature in NIS-Elements software (Nikon, Tokyo, Japan). Greater than 300 cells, imaged from three fields, were analyzed per data point for each experiment.

Walter G.M., Raveh A., Mok S.A., McQuade T.J., Arevang C.J., Schultz P.J., Smith M.C., Asare S., Cruz P.G., Wisen S., Matainaho T., Sherman D.H, & Gestwicki J.E. (2014). High-Throughput Screen of Natural Product Extracts in A Yeast Model of Polyglutamine Proteotoxicity. Chemical biology & drug design, 83(4), 440-449.

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HIV Luciferase Reporter Virus Infection Assay

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HIV luciferase reporter viruses were generated by cotransfection of 293T cells with pNL4-3env-luc, an HIV provirus with env deleted and nef replaced with luciferase, and an Env-expressing plasmid as described (Gorry et al., 2002 (link)). TZM-bl cells were seeded into 96-well plates in media supplemented with 15 μg/ml DEAE-dextran and infected with 10^{4 3}H cpm reverse transcriptase (RT) units of virus stock. Cells were lysed 48 hours post-infection and assayed for luciferase activity. MDM were prepared in 48-well plates and infected overnight with 2 × 10⁴ RT units of virus stock in media supplemented with 2 μg/ml polybrene. Cells were lysed 6 days post-infection and assayed for luciferase activity. Affinofile cells were seeded into 96-well plates and treated for 20 hours with serial dilutions of 3 to 0.09 ng/ml doxycycline (Clontech, Mountain View, CA) to induce CD4 expression and 2 to 0.06 μM Ponasterone A (Invitrogen) to induce CCR5 expression (Johnston et al., 2009 (link)). Cells were then infected with 10⁴ RT units of virus stock, lysed 2 days post-infection, and assayed for luciferase activity. Mock infected cells were used as negative controls. Background luciferase levels were subtracted from all results.

Mefford M.E., Kunstman K., Wolinsky S.M, & Gabuzda D. (2015). Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5. Virology, 481, 210-222.

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Establishing Stable c-Myb-DamID in K562 Cells

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Stable K562 cell lines expressing either 3xFLAG-c-Myb-V5-EcoDam or EcoDam alone were generated by electroporation using the Amaxa Nucleofector system (Lonza Bioscience) with the pINDgw-3xFLAG-c-Myb-V5-EcoDam or pIND-V5-EcoDam, respectively. Following electroporation cell lines were selected with G418 (Invivogen). DamID libraries for EcoDam and c-Myb-V5-EcoDam were made as described in [47 (link)]. In brief: Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) and processed to enrich for DNA methylated by either V5-EcoDam alone or 3xFLAG-c-Myb-V5-EcoDam. Purified DNA was analysed by qPCR using the same amount of DNA for EcoDam and c-Myb-V5-EcoDam [48 (link)] on a Lightcycler480 (Roche). For primers used, see S10 Table. To validate the expression of the full-length 3xFLAG-c-Myb-V5-EcoDam construct, K562 cells were transfected with pINDgw-3xflg-c-Myb-V5-EcoDam or pIND-V5-EcoDam respectively together with the pVgRXR ecdysone receptor-encoding vector. Next day, 2 μM of Ponasterone A (Invitrogen) was added to the cell media and after 24 hours cells were lysed in SDS loading dye and subjected to western blotting on a PVDF membrane with anti-FLAG (Sigma) and anti-GAPDH (Invitrogen) antibodies (S10 Table).

Bengtsen M., Klepper K., Gundersen S., Cuervo I., Drabløs F., Hovig E., Sandve G.K., Gabrielsen O.S, & Eskeland R. (2015). c-Myb Binding Sites in Haematopoietic Chromatin Landscapes. PLoS ONE, 10(7), e0133280.

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Cell Cycle Synchronization and Flow Cytometry

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Cells stably transfected with RNase H1-V5 were seeded in 3 ml of medium at 5 × 10⁵ cells/ml, and expression was induced by the addition of 500 μm CuSO₄. The next day, cells were synchronized in G₁ or G₂, respectively, with 0.5 mm hydroxyurea (Sigma) or 0.5 nm ponasterone A (Invitrogen) or left untreated. After 24 h, 1-ml aliquots of cells from each treatment were used for immunocytochemistry, and an additional 1-ml aliquot from each was pelleted at 1,000 × g_max for 5 min at 4 °C, washed in ice-cold PBS, then stained for 30 min on ice in the dark with 500 μl of propidium iodide staining solution containing 25 μg/ml propidium iodide, 100 μg/ml RNase A, 0.1% sodium citrate, and 0.1% Triton X-100 (Sigma). After 30 min, samples were analyzed by flow cytometry (488-nm excitation, >670-nm emission; FL3). The number of cells was plotted against the DNA content at each time point.

González de Cózar J.M., Gerards M., Teeri E., George J., Dufour E., Jacobs H.T, & Jõers P. (2019). RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA. The Journal of Biological Chemistry, 294(12), 4331-4344.

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Manipulation of Endosomal Trafficking Proteins

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HaCaT, HEK293 and HEK293TT35 (link) were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin-streptomycin (100 U/ml) and glutamine (300 mg/ml). Ecdysone responsive HEK293 cells expressing inducible GFP tagged VPS4 wild type and VPS4EQ mutant were maintained in 400 ug/ml Zeocin and 800 ug/ml G418 and VPS4 expression induced by treating with 1 uM ponA (Ponasterone A, Invitrogen) for 24 h as described previously36 (link). For transient siRNA experiments the cells were transfected using Lipofectamine RNAiMAX (Invitrogen) with siRNAs against TSG101, VPS4A, VPS4B, HRS (ON-TARGETplus SMARTpools, Dharmacon) or non-targeting siRNA (scrambled siRNA) as a control. For HEK293 cell transfections calcium phosphate precipitation was used37.
For HEK293 cell transfections calcium phosphate precipitation was used37 (link).

Broniarczyk J., Pim D., Massimi P., Bergant M., Goździcka-Józefiak A., Crump C, & Banks L. (2017). The VPS4 component of the ESCRT machinery plays an essential role in HPV infectious entry and capsid disassembly. Scientific Reports, 7, 45159.

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Dam-fusion Immunofluorescence Localization

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For immunofluorescence studies of the localization of the Dam-fusions, HeLa cells were grown on poly-lysine coated coverslips in a 24-well chamber and co-transfected with the pIND-(V5)-EcoDam-PP1 isoforms or the pIND-(V5)-EcoDam-PIP-WT/M vectors and the pVgRXR plasmid encoding the Ecdysone and Retinoic X receptors (Invitrogen, Waltham, USA). Twenty hours post transfection, 2 μM of Ponasterone A (Invitrogen) was added and 24 h later, the cells were treated for 4 min at 4°C with ice cold CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM PIPES at pH 6.8) supplemented with 0.2% Triton X-100. Subsequently, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 3% BSA-PBS and incubated first overnight in 1% BSA-PBS with the primary Dam antibody and then with Horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody for 2 h. The HRP signal was enhanced by using the TSA-Plus Fluorescien System (PerkinElmer). DNA was stained with DAPI. The cells were visualized with a Leica TCS SPE laser-scanning confocal system mounted on a Leica DMI 4000B microscope, equipped with a Leica ACS APO 40× 1.30NA oil objective.

Verheyen T., Görnemann J., Verbinnen I., Boens S., Beullens M., Van Eynde A, & Bollen M. (2015). Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits. Nucleic Acids Research, 43(12), 5771-5784.

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