Puromycin dihydrochloride

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Puromycin dihydrochloride is a laboratory reagent used for the selection of eukaryotic cells that have been successfully transfected or transduced with a gene of interest. It functions by inhibiting protein synthesis, allowing for the identification of cells that have integrated the desired genetic material.

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66 protocols using puromycin dihydrochloride

Lentiviral Transduction and Selection

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YSCCC cells were transduced with control lentiviral activation particles (sc-437282; Santa Cruz Biotechnology Inc.) or IFI27 lentiviral activation particles (sc-416981-LAC; Santa Cruz Biotechnology Inc.) according to the manufacturer’s instructions. Three days after transduction, the cells (YSCCC-DNA and YSCCC-IFI27) were selected by incubation with 10 µg/mL puromycin dihydrochloride (sc-108071; Santa Cruz Biotechnology Inc.), 500 µg/mL hygromycin B (sc-29067; Santa Cruz Biotechnology Inc.), and 10 µg/mL Blasticidin S HCl (sc-495389; Santa Cruz Biotechnology Inc.) for at least four generations.

Chiang K.C., Huang S.T., Wu R.C., Huang S.C., Yeh T.S., Chen M.H., Hsu J.T., Chen L.W., Kuo S.F., Chueh H.Y., Juang H.H., Hung S.I., Yeh C.N, & Pang J.H. (2019). Interferon α-inducible protein 27 is an oncogene and highly expressed in cholangiocarcinoma patients with poor survival. Cancer Management and Research, 11, 1893-1905.

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Circadian Rhythm Monitoring in Fibroblasts

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Primary fibroblasts (passage #9–20) were seeded at ~80% confluence onto 35-mm dishes (3 replicate dishes per cell line). Cells were incubated with 8 μg/mL of polybrene (Sigma) diluted in complete media and treated with 10 μL of concentrated pABpuro-BluF virus for ~24 h. Next, the virus was removed, and cells were left in the complete media supplemented with 2 μg/mL puromycin dihydrochloride (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 to 4 days. Following selection with puromycin, cells were synchronized with 0.1 μM dexamethasone (Sigma) diluted in the complete media for 1 h. Prior to placing the dishes into the luminometer, the medium with dexamethasone was changed to the lumicycle medium (no phenol red and 0.1 mM luciferin; Promega, Madison, WI), and the dishes were sealed tightly with high-vacuum silicone grease (Sigma). Real-time luminescence of the control and RP cells was monitored using a LumiCycle luminometer (Actimetrics, Wilmette, IL). The LumiCycle software program (Actimetrics) was used to calculate the period of the luminescence data. Period was determined by analyzing data recorded on days 2 to 5 only. To determine the period length of a given cell line, 3 to 4 independent viral infections were performed (in triplicate).

Shakhmantsir I., Dooley S.J., Kishore S., Chen D., Pierce E., Bennett J, & Sehgal A. (2019). RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation. Journal of biological rhythms, 35(1), 72-83.

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Optimized Cell Culture and Transfection Protocol

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The 293T cells and HeLa Tet-Off TCRβ ter68 (clone 2.2) used in this study are described elsewhere [24 (link)]. They were cultured at 37°C under a 5% carbon dioxide atmosphere in DMEM-F12 (1:1) (Gibco) supplemented with 100 U/mL Penicillin/ 100 μg/mL Streptomycin (Gibco), 10% fetal calf serum (GeneOn), and additives as indicated.
Transfections for NMD depletion and restoration in HeLa TetR TCRβ ter68 (clone 2.2) cells were performed using Lipofectamine 2000 (Invitrogen). Amounts of plasmids needed for exogenous protein expression at near endogenous levels have been titrated. The day after transfection, cells were selected for shRNA expression plasmids carrying the puromycin resistance gene by 1.0 μg/mL puromycin dihydrochloride (Santa Cruz Biotechnology) over two days. Then, cells were exposed to 50 μM biotin (Sigma Aldrich) to boost biotinylation for 16 hours before harvesting.
For the set-up experiments in Fig 1 and for large-scale protein expressions in Figs 3–8, the 293T cells were transfected using Lipofectamine 2000 and expression was allowed for 2 days. Within the last day, cells were exposed to 50 μM biotin for 0, 6, and 16 hours before harvesting as indicated in the results.
For accumulation of hyperphosphorylated proteins such as UPF1, 293T cells were exposed to the phosphatase 2 (PP2) inhibitor okadaic acid at 50 μM for three hours before lysis.

Schweingruber C., Soffientini P., Ruepp M.D., Bachi A, & Mühlemann O. (2016). Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID). PLoS ONE, 11(3), e0150239.

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Lentiviral Knockdown of FOXD2 and FOXD3 in SUDHL4 Cells

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Human B lymphocyte line SUDHL4 was obtained from the American Type Culture Collection (ATCC, CRL-2957TM). The cells were maintained in RPMI-1640, supplemented with 20% fetal bovine serum, 2 mM Glutamine, penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37 °C. Fresh medium was added every two days and the cells were split at the ratio of 1:5. The FOXD2 and FOXD3 shRNA lentiviral particles and control particles were purchased from Santa Cruz Biotechnology. For viral infection, the SUDHL4 cells were placed in 6-well plate at 1 × 106 cells/well supplemented with 8 ug/ml polybrene (Santa Cruz Biotechnology) and 20 ul of either control, FOXD2, or FOXD3 shRNA particles. The cells were centrifuged at 2,000 rpm for 2 hrs at 37 °C. After centrifugation, the cells were returned to humidified incubator for continuing culture. Forty-eight hours later, the cells were split and 0.4 ug/ml puromycin dihydrochloride (Santa Cruz Biotechnology) was added into the cells for selection. The shRNA expression efficiency was determined by quantitative real-time PCR (qPCR).

Batmanov K., Wang W., Bjørås M., Delabie J, & Wang J. (2017). Integrative whole-genome sequence analysis reveals roles of regulatory mutations in BCL6 and BCL2 in follicular lymphoma. Scientific Reports, 7, 7040.

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Generation of IDH1 Knockout Cell Lines

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The CRISPR/Cas9 plasmid products and support reagents including IDH1 CRISPR/Cas9 Knockout (KO) Plasmids, Homology-Directed Repair (HDR) Plasmids, Ultracruz transfection reagent, plasmid transfection medium and puromycin dihydrochloride were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 1.5 × 10⁵ cells/well in 3 mL of antibiotic-free standard growth medium were seeded in 6-well plates, 24h prior to transfection. For each transfection, 2 µg of IDH1 KO plasmids and HDR plasmids were diluted in plasmid transfection medium and mixed with transfection reagent. After a 30 min incubation at room temperature, the mixture was added dropwise to each well. Expression of KO and/or HDR plasmids was visually confirmed by detection of GFP and/or RFP via fluorescent microscopy, 72 h post-transfection. Cells were further selected with 1 µg/mL puromycin for 5 days. After selection, cells with RFP were sorted by a FACS Aria Fusion cell sorter (BD Biosciences, San Jose, CA, USA) and seeded as single cells into 96-well plates. Single-cell colonies were isolated 10 days later followed by numerical expansion and analysis.

Li L., Hu X., Eid J.E., Rosenberg A.E., Wilky B.A., Ban Y., Sun X., Galoian K., DeSalvo J., Yue J., Chen X.S., Blonska M, & Trent J.C. (2020). Mutant IDH1 Depletion Downregulates Integrins and Impairs Chondrosarcoma Growth. Cancers, 12(1), 141.

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RBPJ Knockdown in Cell Lines

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Cells (20 × 10⁴ cells) were seeded into 6‐well plates and exposed to 1 of the 2 RBPJ‐targeting shRNA lentiviral particles (shRBPJ1, sc270318‐v; Santa Cruz Biotechnology; shRBPJ2, Sigma‐Aldrich) or non‐targeting control (sc108080; Santa Cruz Biotechnology) shRNA lentiviral particles. After 3 d of incubation, puromycin dihydrochloride (sc‐108071, Santa Cruz Biotechnology) was used to select stably transduced cells. Puromycin selection was applied for at least 72 h, and RBPJ knockdown was verified by western blotting. RBPJ‐silenced (shRBPJ1 and shRBPJ2) and control (shCONT) cells were expanded for subsequent experiments.

Zhang G., Tanaka S., Jiapaer S., Sabit H., Tamai S., Kinoshita M, & Nakada M. (2020). RBPJ contributes to the malignancy of glioblastoma and induction of proneural‐mesenchymal transition via IL‐6‐STAT3 pathway. Cancer Science, 111(11), 4166-4176.

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Breast Cancer Cell Culture Protocol

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MCF-7/c, MCF-7/SP10+, MDA-MB-231 ctrl and shERβMDA-MB-231 breast cancer cells were cultivated daily in liquified air 95% O₂/5% CO₂ at 37 °C and in DMEM medium, completed with 10% foetal bovine serum (FBS), 1.0 mM pyruvic acid, 2 mM L-glutamine and a cocktail of antibiotics (100 μg/ml of penicillin, 100 μg/ml of gentamicin, 2.5 μg/ml of amphotericin B). Cells passed with 0.05% (w/v) trypsin in PBS, containing 0.02% (w/v) Na₂EDTA. Infections of MCF-7/c cells with shRNA against human ERα or the non-targeted shRNA control and respectively infection against the human gene ERβ for the MDA-MB-231 ctrl, was achieved with the use of Polybrene solution (sc-134220, Santa Cruz Biotechnology, Inc), following the instructions of the manufacturer. For the establishment of the stable clones, puromycin dihydrochloride (0.8 μg/ml) (sc-108071, Santa Cruz Biotechnology, Inc, USA) was added in the medium, and was replaced with fresh puromycin every 3–4 days. DMEM, serum, pyruvate acid, L-glutamine, penicillin, streptomycin, amphotericin B, and gentamicin were supplied by BioseraLTD (Courtaboeuf, France) as previously described4 5 (link).

Karamanou K., Franchi M., Piperigkou Z., Perreau C., Maquart F.X., Vynios D.H, & Brézillon S. (2017). Lumican effectively regulates the estrogen receptors-associated functional properties of breast cancer cells, expression of matrix effectors and epithelial-to-mesenchymal transition. Scientific Reports, 7, 45138.

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Culturing Human Prostate and Breast Cancer Cells

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Human prostate epithelial cells (PrEC; Cambrex Bio Science, Walkersville, MD, USA) and prostate cancer cell lines (PC-3 and LNCaP; DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were cultured as previously described [45 (link)]. The human mammary cancer cell line MDA-MB-453 (DSMZ) was cultured in DMEM (4.5 g/l glucose, w/o L-glutamine and sodium pyruvate; Lonza, Cologne, Germany) supplemented with 20% FCS at 37°C in a humidified atmosphere of 5% CO₂. Transfected LNCaP and MDA-MB-453 cells were cultured in the medium supplemented with 500 mg/ml G418 Sulfate (Thermo Fisher Scientific, Waltham, MA, USA). Transfected PC-3 cells were grown in the medium supplemented with 2.5 mg/ml puromycin dihydrochloride (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). The culture medium was changed every 2 - 3 days. Cells were passaged before reaching confluence using Trypsin/EDTA solution (PromoCell, Heidelberg, Germany). Cell lines were validated using the ATCC cell line authentication service (LGC-ATCC, Middlesex, UK) and were frequently tested for mycoplasma contamination (MycoAlert™ Mycoplasma Detection Kit, Lonza).

Friedemann M., Nacke B., Hagelgans A., Jandeck C., Bechmann N., Ullrich M., Belter B., Neuber C., Sukocheva O., Pietzsch J, & Menschikowski M. (2018). Diverse effects of phospholipase A2 receptor expression on LNCaP and PC-3 prostate cancer cell growth in vitro and in vivo. Oncotarget, 9(89), 35983-35996.

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Molecular Mechanisms in Cancer Cell Lines

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TSA (purity >98%), dimethyl sulfoxide (DMSO) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5,5′,6,6′-tetrachloro-1,1′, 3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Cell nuclear protein extraction kits, MGMT lentiviral activation particles and and control lentiviral activation particles, β-catenin short hairpin RNA (shRNA) lentiviral particle and control shRNA plasmid, Polybrene, and puromycin dihydrochloride were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Li Z.Y., Huang G.D., Chen L., Zhang C., Chen B.D., Li Q.Z., Wang X., Zhang X.J, & Li W.P. (2017). Tanshinone IIA induces apoptosis via inhibition of Wnt/β-catenin/MGMT signaling in AtT-20 cells. Molecular Medicine Reports, 16(5), 5908-5914.

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Apoptosis Regulation Pathway Assay

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Rabbit polyclonal antibodies against cleaved caspase-3, XIAP, pAkt (ser473), pBAD (ser136), Akt1, and actin were purchased from Cell Signaling Technology (Boston, MA, USA). DeadEnd™ Colorimetric TUNEL assay kit was purchased from Promega (Madison, WI, USA). XIAP siRNA (Catalog ID: 4390824) and siRNA negative control (Catalog ID: 4390843) were purchased from Life Technologies (Grand Island, NY, USA). Akt1 shRNA lentiviral particles (Catalog ID: 29195 v), polybrene, and puromycin dihydrochloride were purchased from Santa Cruz Biotechnologies (Dallas, TX, USA).

Jain S, & Jani A. (2020). Renal Tubular Cells from Hibernating Squirrels are Protected against Cisplatin Induced Apoptosis. International Journal of Nephrology, 2020, 6313749.

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