The siRNA targeted to GL4 luciferase (Sense strand: 5'- GGACGAGGACGAGC-ACUUCUU-3', Antisense strand: 3'-UUCCUGCUCCUGCUCGUGAAG-5') was custom synthesized (Thermo Fisher Scientific, Carlsbad, CA). Negative control (NC ) siRNA (Silencer® Negative Control No. 1 siRNA) and the siRNA targeted to mouse EGFR gene (silencer® s65374, EGFR siRNA) were ordered from Thermo Fisher Scientific. Murine squamous cell carcinoma (SCC-VII) cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS ) and 2% penicillin/streptomycin. SCC-VII cells stably expressing luciferase (SCC-VII-Luc ) were generated by transfecting SCC-VII cells with Cignal Lenti signal transducer and transcription activator 3 (STAT3) Reporter (luc) Kit (Qiagen, Valencia, CA) which contains a VSV-g pseudo typed lentivirus particle expressing the firefly luciferase gene under the control of a CMV promoter and the tandem repeats of STAT3 transcriptional response element. The lentivirus transfected SCC-VII cells were selected with 0.75 µg/mL puromycin (MP biomedicals, Santa Ana, CA) and maintained in RPMI-1640 medium supplemented with 10% FBS, 2% penicillin/streptomycin and 0.75 µg/mL puromycin. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. All cell culture products were obtained from Lonza (Allendale, NJ).
Puromycin
Manufactured by MP Biomedicals
Sourced in United States
Puromycin is a laboratory reagent used as a selective antibiotic for the establishment of stable cell lines. It functions by inhibiting protein synthesis, which allows for the selection of cells that have been successfully transfected with a puromycin resistance gene.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Corning (4 mentions)
Prsv rev 12253, by Addgene (3 mentions)
Pmdg 2 12259, by Addgene (3 mentions)
Pmdlg prre 12251, by Addgene (3 mentions)
Dharmafect transfection reagent, by Thermo Fisher Scientific (3 mentions)
Doxycycline, by Merck Group (3 mentions)
Polybrene, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (3 mentions)
Durapore pvdf membrane, by Merck Group (3 mentions)
Viral particles, by GE Healthcare (2 mentions)
R5020, by Merck Group (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Geneticin, by Lonza (2 mentions)
Geneticin, by Thermo Fisher Scientific (2 mentions)
Facsariaii, by BD (2 mentions)
Fugene hd reagent, by Promega (2 mentions)
Fugene hd, by Promega (2 mentions)
Puromycin, by Corning (2 mentions)
47 protocols using puromycin
1
Establishment of SCC-VII-Luc Cell Line
2
Puromycin Labeling and Proteome Enrichment
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Puromycin labeling was performed as previously described.72 (link) Briefly, cells were incubated in Puromycin (MP Biomedicals, 0210055210) containing media (10 μg/mL) for 20 min, washed twice with cold PBS, lysed in 1x RIPA buffer (Thermo Scientific, J62725AP) containing proteinase inhibitors and phosphatase inhibitors. 50–100 μg of protein from cell lysate was incubated at 4°C overnight with 1 μg of anti-Puromycin antibody (MilliporeSigma, Clone: 12D10). Next, Puromycin labeled proteins were purified by MS-Compatible Magnetic IP Kit (Thermo Scientific, 90409) following the manufacturer’s instruction, and processed for mass spectrometry analysis.
3
Transient and Stable Transfection Protocols
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For transient transfection, HEK293T cells were cultured in an antibiotic‐free medium at 60% convergence. Plasmids containing Opti‐MEM (31985070, Gibco) was gently mixed and incubated with Lipofectamine 2000 (11668019, Invitrogen) containing Opti‐MEM at room temperature for 20 min. The mixture was dropped into HEK293T cells for indicated time. The transfected cells were used for functional experiments.
For stable transfection, lentivirus was first produced with HEK293T cells cotransfected with the lentiviral constructs, packaging plasmid (psPAX2) and envelope plasmid (pMD2.G) using Lipofectamine 2000 with the ratio 4:3:1. Virus was collected, filtered, and added to KG1A cells with polybrene (HY‐112735, MCE) for 24 h. The infected KG1A cells were further selected with 1 µg mL−1 puromycin (0219453925, MP) for 2 weeks to get stable transfected cell lines.
For stable transfection, lentivirus was first produced with HEK293T cells cotransfected with the lentiviral constructs, packaging plasmid (psPAX2) and envelope plasmid (pMD2.G) using Lipofectamine 2000 with the ratio 4:3:1. Virus was collected, filtered, and added to KG1A cells with polybrene (HY‐112735, MCE) for 24 h. The infected KG1A cells were further selected with 1 µg mL−1 puromycin (0219453925, MP) for 2 weeks to get stable transfected cell lines.
4
T47D Cell Line Manipulation and Prolactin Response
5
Lentiviral Overexpression and Knockdown of BAP31 in Liver Cancer Cells
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Full-length BAP31 cDNA (NCBI Reference Sequence: NM_005745.7) was cloned into the pCDH-CMV-MCS-EF1-GFP-Puro vector. The GFP-BAP31 lentivirus and vector control were constructed by GeneCreate Co., Ltd. (Wuhan, China). BAP31-specific shRNA (GGTGAACCTCCAGAACAAT) was inserted into the hU6-MCS-Ubiquitin-EGFP-IRES-Puro vector. The BAP31-shRNA lentivirus and vector control were constructed by GeneChem Co., Ltd. (Shanghai, China).
Hep3b and MHCC97h cells were seeded in 96-well plates. After 24 h, 10 μl of virus [diluted in enhanced infection solution (ENi.S.), 1 × 108 TU/ml] and 10 μl of polybrene (E) (diluted polybrene in ENi.S., 50 μg/ml) was added to 80 μl of ENi.S. per well. After 12 h, the infection solution was removed and replaced with fresh medium containing 10% FBS. Puromycin (5 μg/ml) (MP Biomedicals, Shanghai, China) was added into the supernatant to select transfected cells. BAP31 expression was validated by qPCR and western blot.
Hep3b and MHCC97h cells were seeded in 96-well plates. After 24 h, 10 μl of virus [diluted in enhanced infection solution (ENi.S.), 1 × 108 TU/ml] and 10 μl of polybrene (E) (diluted polybrene in ENi.S., 50 μg/ml) was added to 80 μl of ENi.S. per well. After 12 h, the infection solution was removed and replaced with fresh medium containing 10% FBS. Puromycin (5 μg/ml) (MP Biomedicals, Shanghai, China) was added into the supernatant to select transfected cells. BAP31 expression was validated by qPCR and western blot.
6
Characterization of Murine Ovarian Cancer ID8 Cell Line
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ID8 is a cell line derived from mouse ovarian surface epithelial cells that have the ability to form extensive peritoneal tumors in vivo and have no known genetic mutations [24 (link)]. ID8 cells have variable ability to perform oxidative phosphorylation and glycolysis (glutamine independent) [25 (link)]. The ID8 cells used in this study were a gift from Dr. Vince Tuohy.
ID8 (syngeneic) EOC cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% heat-inactivated FBS (Atlas Biologicals Cat # F-0500-D, Lot F31E18D1), which were grown under standard conditions. For luciferase transduction, in short, HEK293T/17 (ATCC CRL-11268) cells were plated and co-transfected with Lipofectamine 3000 (L3000015 Invitrogen, Waltham, MA, USA), third-generation packaging vectors pRSV-REV #12253, pMDG.2 #12259, and pMDLg/pRRE #12251 (Addgene, Watertown, MA, USA), and a lentiviral vector directing the expression of luciferase reporter pHIV, Luciferase #21375, at 4.5 µg (Addgene, Watertown, MA, USA). Viral particles were harvested, filtered through a 0.45 µm Durapore PVDF Membrane (Millipore Sigma, St. Louis, MO, USA), and added to each cell line’s culture medium. Viral infections were carried out over 72 h and the transduced cells were selected based on their resistance to 2 μg/mL of puromycin (MP Biomedicals, Santa Ana, CA, USA) [26 (link)].
ID8 (syngeneic) EOC cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% heat-inactivated FBS (Atlas Biologicals Cat # F-0500-D, Lot F31E18D1), which were grown under standard conditions. For luciferase transduction, in short, HEK293T/17 (ATCC CRL-11268) cells were plated and co-transfected with Lipofectamine 3000 (L3000015 Invitrogen, Waltham, MA, USA), third-generation packaging vectors pRSV-REV #12253, pMDG.2 #12259, and pMDLg/pRRE #12251 (Addgene, Watertown, MA, USA), and a lentiviral vector directing the expression of luciferase reporter pHIV, Luciferase #21375, at 4.5 µg (Addgene, Watertown, MA, USA). Viral particles were harvested, filtered through a 0.45 µm Durapore PVDF Membrane (Millipore Sigma, St. Louis, MO, USA), and added to each cell line’s culture medium. Viral infections were carried out over 72 h and the transduced cells were selected based on their resistance to 2 μg/mL of puromycin (MP Biomedicals, Santa Ana, CA, USA) [26 (link)].
7
Lentiviral Transduction and Cell Selection
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A549, PLA-801D and GLC-82 cells were seeded in 96-well plates. After 24 h, 10 μL of virus (diluted in enhanced infection solution [ENi.S.], 1 × 107 TU/mL) was added to 80 μL of ENi.S. and 10 μL of polybrene (E) (diluted polybrene in ENi.S., 50 μg/mL). After 12 h, the infection solution was changed to fresh nutrient medium. Puromycin (5 μg/mL) (MP Biomedicals, Shanghai, China) was added into the supernatant for 1 week to select for transfected cells. GLC-82 cell lines with stably up-regulated BCAP31 can’t established.
8
Knockdown and Overexpression of AQP3 in HTR8/SVneo Cells
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Plasmids containing AQP3 knockdown, AQP3 overexpression, or their respective negative control plasmids were purchased from Weijiang Biotechnology (China). Lentiviral packaging was performed in 293 T cells according to the manufacturer’s instructions (Suzhou GenePharma, China). Lentivirus in the supernatant was collected to transfect cells. Controls were transfected with empty vector. Cells were divided into four groups: interfering AQP3 (AQP3-shRNA) group, interfering empty vector (CON-shRNA) group, overexpressing AQP3 (AQP3-OE) group and overexpressing empty vector (CON-OE) group. Cells were infected with viruses at the following multiplicities of infection (MOI): AQP3-shRNA (MOI =100); CON-shRNA (MOI =100); AQP3-OE (MOI =150); CON-OE (MOI =100). Strict phenotype selection was performed on stably infected HTR8/SVneo cells with 0–10 μg/mL puromycin (MPbio, USA) to use resistance as a screening index. Furthermore, cells were stably cloned in 0.5 μg/mL puromycin.
9
CRISPR Screen for Alisertib Sensitivity in MDA-MB-231 Cells
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Lentiviral libraries were produced by transfecting Human CRISPR Knockout Pooled Library (GeCKO v2 A and B, Addgene, cat#1000000048) plasmids with pMD2.G and psPAX2 plasmids using PEI (Sigma). MDA-MB-231 cells were infected with the viral library with an MOI of 0.3 and selected with puromycin (MP Biomedicals) for 7 days. Cells were then divided into three groups: directly harvested, DMSO treated for 7 days, and alisertib treated for 7 days. For each group, the number of cells were guaranteed to be over 4 × 107 to achieve 300 × coverage. Genomic DNA was extracted using an HP Tissue DNA Midi Kit (OMEGA, cat#D5197). The sgRNA cassettes were amplified to construct the libraries. The libraries were sequenced on an Illumina HiSeq2000 (150 bp, paired-end). The data was analyzed through the MAGeCK package [12 (link)].
10
Lentiviral-based overexpression and knockdown of PHF2 and CDKN2B-AS1 in EBV+ cell lines
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The lentiviruses for PHF2 and CDKN2B-AS1 overexpression and the corresponding control viruses were purchased from VectorBuilder (China). C666-1 and HK1 EBV+ cell lines were infected with recombinant lentivirus plus 5 mg/mL polybrene (VectorBuilder, China) according to the manufacturer’s instructions and were selected using 5 μg/mL puromycin (MP Biomedicals, LLC, USA).
For PHF2 knockdown, siRNA duplexes targeting PHF2 and the negative control siRNA (si-NC) were designed and synthesized by RiboBio (Guangzhou, China). For CDKN2B-AS1 knockdown, the lncRNA smart silencer (a mixture of three siRNAs and three ASOs targeting CDKN2B-AS1) and smart silencer of the negative control (ss-NC) was used (RiboBio, Guangzhou, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection according to the manufacturer’s instructions. The sequences of siRNA and lncRNA smart silencer are shown inTable S5 . To validate the effects of overexpression and knockdown of target genes in C666-1 and HK1 EBV+ cell lines, the changes of protein abundance of PHF2 were verified by western blot. For the non-coding RNA CDKN2B-AS1, the expression changes were measured by qPCR.
For PHF2 knockdown, siRNA duplexes targeting PHF2 and the negative control siRNA (si-NC) were designed and synthesized by RiboBio (Guangzhou, China). For CDKN2B-AS1 knockdown, the lncRNA smart silencer (a mixture of three siRNAs and three ASOs targeting CDKN2B-AS1) and smart silencer of the negative control (ss-NC) was used (RiboBio, Guangzhou, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection according to the manufacturer’s instructions. The sequences of siRNA and lncRNA smart silencer are shown in
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