Puromycin

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Puromycin is a laboratory reagent used for selection of mammalian cells expressing a puromycin resistance gene. It acts as an antibiotic that inhibits protein synthesis, leading to cell death in cells that do not express the resistance gene.

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O-propargyl-puromycin (5 citations) Puromycin medium (4 citations) Puromycin (#ant-pr-1 (3 citations) Puromycin resistance (3 citations) Puromycin and trizol reagent (2 citations) Puromycin for shRNA (2 citations) Puromycin-resistant feeder fibroblasts (2 citations) Puromycin solution (2 citations) Linearized pcDNA3.1/puromycin (2 citations) OP-Puromycin protein translation kit (2 citations)

3 137 protocols using puromycin

Generating ERBB4 Overexpression Cell Lines

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To generate Ba/F3 and NIH-3T3 cell lines with stable overexpression of ERBB4 variants, Phoenix-Ampho packaging cells were transfected with retroviral pBABEpuro-gateway constructs encoding ERBB4 variants or enhanced GFP (eGFP) using FuGENE 6 transfection reagent (Promega) according to manufacturer's instructions. The retroviral supernatants of Phoenix-Ampho cells harvested 24 and 48 hours after transfection were incubated on Ba/F3 and NIH-3T3 cells for 6 hours on 2 consecutive days in the presence of 0.8 µg/mL hexadimethrine bromide (Polybrene, Sigma-Aldrich). Cell pools with stable expression were selected with 2 µg/mL puromycin (Gibco) and then maintained in 1 µg/mL puromycin for Ba/F3 cells, or with 6 µg/mL puromycin and then maintained in 3 µg/mL puromycin for NIH-3T3 cells.
To generate BEAS-2B cell lines with stable overexpression of ERBB4 variants, Platinum-E packaging cells were transfected with retroviral pMSCV-PGK-Puro-IRES-GFP constructs encoding ERBB4 variants or an empty vector using X-tremeGENE 9 transfection reagent (Roche) according to manufacturer's instructions. The retroviral supernatants of Platinum-E cells were harvested 48 hours after transfection and incubated on BEAS-2B cells for 72 hours. Transduced BEAS-2B cells were cultured in the presence of 1 µg/mL puromycin to select and maintain cell lines with stable expression.

Chakroborty D., Ojala V.K., Knittle A.M., Drexler J., Tamirat M.Z., Ruzicka R., Bosch K., Woertl J., Schmittner S., Elo L.L., Johnson M.S., Kurppa K.J., Solca F, & Elenius K. (2022). An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations. Cancer Research Communications, 2(1), 10-27.

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Culturing MEFs, T98G, HEK293T, and NIH 3T3 Cells

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MEFs (both males and females) were cultured in DMEM (Cellgro), 10% fetal bovine serum, 1% Penicillin/Streptomycin (Cellgro), 2.5 μg/mL Puromycin (GIBCO), and 50 μg/mL Hygromycin B (Invitrogen). T98G cells (glioblastoma cells from a male individual, obtained from ATCC) were cultured in DMEM (Cellgro), 10% fetal bovine serum, and 1% Penicillin/Streptomycin (Cellgro). T98G cells with RNAi were cultured in DMEM (Cellgro), 10% fetal bovine serum, 1% Penicillin/Streptomycin (Cellgro), and 1 μg/mL Puromycin (GIBCO). T98G cells engineered with the FUCCI probes and pTRIPZ-EMI1-FLAG were cultured in DMEM (Cellgro), 10% fetal bovine serum, 1% Penicillin/Streptomycin (Cellgro), 1 μg/mL Puromycin (GIBCO), 150 μg/mL Hygromycin B (Invitrogen), and 10 μg/mL Blasticidin (Invivogen). HEK293T cells (from a female fetus) cultured in DMEM (Cellgro), 10% donor calf serum, and 1% Penicillin/Streptomycin (Cellgro). NIH 3T3 cells were cultured in DMEM (Cellgro), 10% fetal bovine serum, 1% Penicillin/Streptomycin (Cellgro), and 2.5 μg/mL Puromycin (GIBCO). 0.5 μg/mL doxycycline was the working concentration for all doxycycline systems. All cultures were maintained in 5% CO₂ at 37°C. Regarding mice use for the generation of MEFs, all animal work was approved by the NYULMC Institutional Animal Care & Use committee.

Bainor A.J., Saini S., Calderon A., Casado-Polanco R., Giner-Ramirez B., Moncada C., Cantor D.J., Ernlund A., Litovchick L, & David G. (2018). The HDAC-Associated Sin3B Protein Represses DREAM Complex Targets and Cooperates with APC/C to Promote Quiescence. Cell reports, 25(10), 2797-2807.e8.

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Culturing MEFs, T98G, HEK293T, and NIH 3T3 Cells

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Lentiviral Transduction and Puromycin Selection

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Target HaCaT and PDV cells were seeded at 1 × 10⁵ cells per well in cDMEM in a 6 well plate. After 24 h, media was replaced with 500 µl of lentivirus and 500 µl cDMEM. Media was changed the following day and remained on cells for a further 24 h. HaCaT cells were then selected with 1 μg/ml puromycin (Gibco, Invitrogen), while PDV cells were selected with 1.1 μg/ml puromycin (54), chosen based on the results of a puromycin kill curve. Surviving cells were expanded and used to generate microparticles.

Zhang J., Burn C., Young K., Wilson M., Ly K., Budhwani M., Tschirley A., Braithwaite A., Baird M, & Hibma M. (2018). Microparticles produced by human papillomavirus type 16 E7-expressing cells impair antigen presenting cell function and the cytotoxic T cell response. Scientific Reports, 8, 2373.

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Parkin Overexpression in Neuroblastoma Cells

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Neuroblastoma (SH-SY5Y) cells stably overexpressing Parkin were generated using lentiviral particles. To produce Parkin-expressing lentiviral particles, a cassette consisting of the open reading frame of the Parkin gene (NM_004562.3), the IRES sequence, and the Puromycin resistance gene were cloned into pLenti4/V5-DEST (ThermoFisher) plasmid (pLenti4-TH-IRES-Puromycin). Next, 293FT cells were cotransfected with pLenti-Parkin-IRES-Puromycin and the ViraPowerTM Packaging Mix (ThermoFisher) to generate a lentiviral stock. SH-SY5Y cells were transduced using lentiviral particles for 48 h and subsequently selected by using 2 μg/mL Puromycin (ThermoFisher) for 48 h.

Grossmann D., Berenguer-Escuder C., Bellet M.E., Scheibner D., Bohler J., Massart F., Rapaport D., Skupin A., Fouquier d'Hérouël A., Sharma M., Ghelfi J., Raković A., Lichtner P., Antony P., Glaab E., May P., Dimmer K.S., Fitzgerald J.C., Grünewald A, & Krüger R. (2019). Mutations in RHOT1 Disrupt Endoplasmic Reticulum–Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease. Antioxidants & Redox Signaling, 31(16), 1213-1234.

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Lentiviral Transduction of NEPC Organoids

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We performed lentiviral short hairpin (shRNA) transduction of the NEPC patient-derived organoid (PDO) WMC154 with lentivirus packaged with shRNA targeting CELSR3 (targeting sequence in the 3′ untranslated region) or scrambled sequence as a control as we have described previously (31 (link)). Briefly, NEPC PDO WMC154 were dissociated with TrypLE (Gibco) and resuspended in organoid medium (38 (link)) containing Polybrene (Millipore) and Y27632 (Selleckchem, S1049). The dissociated organoid cells were combined with viral suspension and centrifugated at 600 × g, 32°C for 60 minutes. The organoid/virus mix was then incubated at 37°C overnight. Organoid cells were subsequently collected, resuspended in 120 μL of Matrigel (Corning) and seeded in a 24-well plate. Antibiotic selection was performed using 1 μg/mL puromycin (Thermo Fisher Scientific) for 7 days. LNCaP-N-Myc cells were transduced in 6-well plates in medium containing Polybrene (Millipore). Cells were selected with 2 μg/mL puromycin (Thermo Fisher Scientific) for 7 days and grown without puromycin 1 for 6 weeks.

Van Emmenis L., Ku S.Y., Gayvert K., Branch J.R., Brady N.J., Basu S., Russell M., Cyrta J., Vosoughi A., Sailer V., Alnajar H., Dardenne E., Koumis E., Puca L., Robinson B.D., Feldkamp M.D., Winkis A., Majewski N., Rupnow B., Gottardis M.M., Elemento O., Rubin M.A., Beltran H, & Rickman D.S. (2023). The Identification of CELSR3 and Other Potential Cell Surface Targets in Neuroendocrine Prostate Cancer. Cancer Research Communications, 3(8), 1447-1459.

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Retroviral Transduction of BaF3 Cells with FGFR3

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Murine pro-B lymphocyte BaF3 cells (DSMZ, Germany) were transduced with retroviral particles to stably overexpress either human FGFR3 IIIc or FGFR3 IIIc G380R. Briefly, GP-2 HEK packaging cell line (Clontech) was co-transfected with a pantropic VSVg vector (Clontech) and a pBABE-puro-hFGFR3 IIIc or hFGFR3 IIIc G380R (GeneArt, Thermo Fisher Scientific) using a standard CaCl2 procedure. After 48h, supernatant containing retroviral particles were harvested and viral RNA copy was determined by RT-qPCR (Retro-X™ qRT-PCR Titration Kit, Clontech).
BaF3 were then transduced by spinocculation (centrifugation 800g, 30 minutes at 32°C) and puromycin (Gibco) was added 3 days later to start the selection. 2μg/mL final puromycin concentration was chosen based on a puromycin kill curve made with non-transfected BaF3.
BaF3 mutant cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco), 1% Penicillin/Streptomycin/Glutamine (Gibco), 10ng/mL recombinant murine IL-3 (R&D Systems) and 2μg/mL puromycin.

Gonçalves D., Rignol G., Dellugat P., Hartmann G., Sarrazy Garcia S., Stavenhagen J., Santarelli L., Gouze E, & Czech C. (2020). In vitro and in vivo characterization of Recifercept, a soluble fibroblast growth factor receptor 3, as treatment for achondroplasia. PLoS ONE, 15(12), e0244368.

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Lentiviral Knockdown of DVL-1 in MDA-MB-231 Cells

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MDA-MB-231 were infected by pLKO.1-puro based shRNA MISSION lentiviral transduction particles for DVL-1 (TRCN0000441114, Sigma), and Non-Target shRNA control transduction particles (SHC002V, Sigma). 24 h prior to transduction, cells were plated in order to reach 80% confluency at the time of transduction. The transduction was enhanced with Hexadimethrine Bromide (Sigma, H9268) at a final concentration of 8 μg/ml. Following the addition of Hexadimethrine Bromide, the appropriate amount of viral particles were added at 2× multiplicity of infection (MOI) to the media, which was replaced with fresh media after 24 h. The puromycin selection was started 48 h after transduction at a concentration 1 μg/ml of puromycin (Gibco, A11138-03), and the puromycin-containing media was replaced every 3–4 days until total selection was achieved.

Sharma M., Castro-Piedras I., Rodgers A.D, & Pruitt K. (2021). Genomic profiling of DVL-1 and its nuclear role as a transcriptional regulator in triple negative breast cancer. Genes & Cancer, 12, 77-95.

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Immortalized Fibroblasts with Induced HOTAIR Expression

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Fibroblasts were grown from healthy control forearm biopsies and immortalised using retrovirus expressing human telomerase (hTERT) as previously outlined [3 (link)]. HOTAIR expression was then induced by transduction with GIPZ lentiviruses carrying HOTAIR gene sequence or scrambled RNA sequence as control in frame with puromycin resistance gene and GFP fluorochrome gene (Open Biosystems, Surrey, UK). For this purpose, cells were seeded at 50% confluence and infected with lentiviral particles in serum-free DMEM and incubated for 6 h, after which an additional 1 ml of DMEM containing 10% FCS was added and the cells were incubated for a further 72 h. Stably transduced cells were positively sorted for GFP fluorescence employing fluorescence-activated cell sorting in sterile conditions (BD INFLUX). Positively sorted cells were further selected in media containing 1.0 μg/ml puromycin (Life Technologies) for 10 days.

Wasson C.W., Ross R.L., Wells R., Corinaldesi C., Georgiou I.C., Riobo-Del Galdo N.A, & Del Galdo F. (2020). Long non-coding RNA HOTAIR induces GLI2 expression through Notch signalling in systemic sclerosis dermal fibroblasts. Arthritis Research & Therapy, 22, 286.

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Generating FTO Knockout Cell Lines

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Fto^−/− MEFs was a kind gift from Dr. Giles Yeo (University of Cambridge) [5] (link) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1x essential amino acids (Invitrogen) and 50 µM β-mercaptoethanol. They were derived from global germline Fto knockout mice described in F. McMurray et al. [9] (link). Stable HEK293T FTO knockout and control cells were established by puromycin selection after transduction of purified lentiviral FTO or non-silencing shRNAmir particles (OpenBiosystems) into HEK293T cells. Stable HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 0.5 µg/ml puromycin (Invitrogen). MEFs and stable HEK293t were treated with WNT3a or WNT5a conditioned medium obtained from mouse L cells stably expressing either Wnt3a or Wnt5a (ATCC) for the times indicated in figure legends. Control medium was from L cells.

Osborn D.P., Roccasecca R.M., McMurray F., Hernandez-Hernandez V., Mukherjee S., Barroso I., Stemple D., Cox R., Beales P.L, & Christou-Savina S. (2014). Loss of FTO Antagonises Wnt Signaling and Leads to Developmental Defects Associated with Ciliopathies. PLoS ONE, 9(2), e87662.

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