Transfection of LentiX cells with shRNAs against LacZ and Mcm6 (pLKO lentiviral vector TRCN0000124142) followed by infection of CH12 cells with lentiviral supernatants was done exactly as described (33, 43) . In brief, 24 h after spininfection (2,350 rpm for 90 min), cells were selected with 1 g/ml Puromycin (Roche) and activated with IL-4, anti-CD40 and TGF-β 24 h later in the presence of Puromycin. Cells were harvested 48 h after activation. For AIDER expression, the pMX retroviral vector expressing AIDER-IRES-mCherry was transfected into PlatE cells and the viral supernatants (48 h or 72 h) were used to spin-infect CH12 cells for 90 min at 2,350 rpm. AIDER-positive cells were sorted based on mCherry expression. These cells were then infected with shRNAs and activated, as described above. To trigger AIDER nuclear import, 2 M 4-hydroxy tamoxifen (4-HT; Sigma) was added at the time of activation.
Puromycin
Manufactured by Roche
Sourced in United States, Germany, Switzerland
Puromycin is a laboratory reagent used in cell culture experiments. It functions as an antibiotic that inhibits protein synthesis in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Polybrene, by Merck Group (3 mentions)
4 hydroxytamoxifen 4 ht, by Merck Group (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hygromycin, by Roche (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Flag antibody, by Merck Group (1 mentions)
Fcas aria 2, by BD (1 mentions)
Zeocin, by Roche (1 mentions)
Lenticrisprv2gfp, by Addgene (1 mentions)
Cycloheximide (chx), by Roche (1 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Puromycin, by MP Biomedicals (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Plko 1 puro plasmid vector, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Mia paca 2, by Genechem (1 mentions)
Mia paca 2, by Thermo Fisher Scientific (1 mentions)
15 protocols using puromycin
1
Lentiviral Knockdown and AIDER-Induced Activation in CH12 Cells
2
Generation of Inducible Cell Lines for Molecular Studies
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The human embryonic kidney cell line HEK-293 T (293 T), the Madin Darby Canine Kidney (MDCK) cell line, the v-Ha-Ras stable transformed MDCK (21D1) cell line have all been previously described [32 (link), 34 (link)]. To generate the doxycycline inducible SPSB1 cell line in 21D1 cells, a tetracycline-inducible vector, pTRE was utilized [36 (link)]. Briefly, pTRE-FLAG-SPSB1 and pEFpurop-Tet-on [36 (link)] were co-transfected into 21D1 cells by using FuGENE HD transfection reagent (Roche, Basel, Switzerland) following the manufacturer’s instructions and selected for using puromycin (Roche, Basel, Switzerland). To generate the doxycycline inducible v-Ha-Ras cell line in MDCK cells, pTRE-v-Ha-Ras and pEFpurop-Tet-on were co-transfected into MDCK cells by using FuGENE 6 transfection reagent (Roche, Basel, Switzerland) following the manufacturer’s instructions and selected for using puromycin (Roche, Basel, Switzerland). All positive clones were selected by Western blot analysis using FLAG antibody (Sigma-Aldrich) or Ras antibody (In house made). All cells were maintained in Dulbecco’s Modified Eagle’s Medium contained 10% foetal bovine serum (FBS) (DKSH, Hallam, Victoria, Australia), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptoMYCin (Invitrogen).
3
Lentiviral Knockdown and AIDER-Induced Activation in CH12 Cells
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Transfection of LentiX cells with shRNAs against LacZ and Mcm6 (pLKO lentiviral vector TRCN0000124142) followed by infection of CH12 cells with lentiviral supernatants was done exactly as described (33, 43) . In brief, 24 h after spininfection (2,350 rpm for 90 min), cells were selected with 1 g/ml Puromycin (Roche) and activated with IL-4, anti-CD40 and TGF-β 24 h later in the presence of Puromycin. Cells were harvested 48 h after activation. For AIDER expression, the pMX retroviral vector expressing AIDER-IRES-mCherry was transfected into PlatE cells and the viral supernatants (48 h or 72 h) were used to spin-infect CH12 cells for 90 min at 2,350 rpm. AIDER-positive cells were sorted based on mCherry expression. These cells were then infected with shRNAs and activated, as described above. To trigger AIDER nuclear import, 2 M 4-hydroxy tamoxifen (4-HT; Sigma) was added at the time of activation.
4
Generating CRISPR Knockout Clones
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For gene deletion, a pair of single guide RNAs (sgRNAs) were designed with the CRISPOR program. One plasmid expressing both gRNA, Cas9 and green fluorescent protein (LentiCRISPRV2GFP, Plasmid # 82416 Addgene) was nucleofected into Ramos cells using Amaxa (Lozano) according to the manufacturer’s protocol. At 24 h after transfection, GFP cells were sorted with BD FCAS Aria II and plated into single clones in 96-well plates. Individual clones were genotyped by PCR to identify mutated clones by insertion or deletion. Candidate clones were further confirmed by Sanger sequencing and western blot. Guide RNA sequences: pol eta gRNAfor: 5’-GGTGAGGTTAGCTTTCCCAC-3’ and pol eta gRNARev: 5’-GTGGGAAAGCTAACCT-CACC-3’, AICDA gRNAfor: 5’-GTGGAATTGCTCTTCCTCC-3’, AICDA gRNARev: 5’-GGAGGAAGAG CAATTCCAC-3’. A vector expressing full-length human polη was described previously (27 (link)), and a vector expressing full-length human AICDA was described in (21 (link)) and used for complementation of the KO cell lines. polη Zeocin- and AID puromycin-resistant clones were selected with 150 μg/mL Zeocin (Roche, Mannheim, Germany) and 600 ng/mL puromycin (Invitrogen), respectively.
5
RIP Assay for RNA-Binding Proteins
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RIP experiments were performed using a modified protocol from prior reports28 ,29 . HeLa cells were transfected with the indicated constructs and 24 h post-transfection, cells were treated with cycloheximide (100 μg/ml; Sigma Aldrich) or puromycin (50 μg/ml; MP biomedicals) for 15 or 30 min, respectively. The cells were washed twice with PBS and scraped in 500 μl RIP lysis buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5% (v/v) NP-40) supplemented with 40 U/ml SUPERase•In RNase Inhibitor (Invitrogen), EDTA-free protease inhibitor (Roche) and cycloheximide or puromycin.
The crude extracts were incubated on ice for 40 min and sonicated on ice for 1 min (1s on, 2s off). The extracts were cleared by centrifugation at max speed for 15 min, and 10% of the input was saved for RNA extraction. 10 μl of equilibrated GFP-Trap beads was added to the extract and incubated at 4 °C for 2 h. The beads were washed 4 times for 10 min total with high salt wash buffer (20 mM HEPES pH 7.5, 350 mM KCl, 10 mM MgCl2, 0.1% (v/v) NP-40) supplemented with SUPERase•In RNase Inhibitor and cycloheximide or puromycin. RNA was extracted from each input and bead sample using TRI Reagent. 1.5 μl of purified RIP-RNA and input RNA samples were used for cDNA synthesis. Enrichment relative to input RNA was calculated using the formula 100 × 2[(Cp (Input) – 4.907) – Cp (IP)] and expressed as “% input RNA”.
The crude extracts were incubated on ice for 40 min and sonicated on ice for 1 min (1s on, 2s off). The extracts were cleared by centrifugation at max speed for 15 min, and 10% of the input was saved for RNA extraction. 10 μl of equilibrated GFP-Trap beads was added to the extract and incubated at 4 °C for 2 h. The beads were washed 4 times for 10 min total with high salt wash buffer (20 mM HEPES pH 7.5, 350 mM KCl, 10 mM MgCl2, 0.1% (v/v) NP-40) supplemented with SUPERase•In RNase Inhibitor and cycloheximide or puromycin. RNA was extracted from each input and bead sample using TRI Reagent. 1.5 μl of purified RIP-RNA and input RNA samples were used for cDNA synthesis. Enrichment relative to input RNA was calculated using the formula 100 × 2[(Cp (Input) – 4.907) – Cp (IP)] and expressed as “% input RNA”.
6
Engineered TREX-293 Cell Lines
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The TREX-293 cell line was a gift from J. Niles (MIT, Cambridge, MA). TREX-293 cells were seeded at 75% confluence and cotransfected with expression vector pcDNA-LIC-GRA15II-HF or ROP38I-HF and a puromycin resistance vector (ratio of 10:1), using the X-tremeGENE 9 DNA transfection reagent (Roche). Cells were split 2 days posttransfection and subjected to puromycin (Calbiochem) selection at 1 μg/ml. Foci were picked and expanded at least 1 week postselection, and positive foci were selected through HA expression using immunofluorescence and immunoblotting.
7
Fascin Knockdown in TNBC Cells
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Human TNBC MDA-MB-231 cells (American Type Culture Collection), were cultured at 37°C for 24 h with 5% CO2 in DMEM (Sigma-Akdrech, Merck KGaA) with 10% fetal bovine serum (Biosera France SAS). Following recombination of the short hairpin RNA (shRNA) against fascin, the pLKO.1-puro plasmid vector (1 µg/µl; Clone ID: NM_003088.2-1699s1c; Sigma-Aldrich, Merck KGaA), containing the puromycin-resistance gene, was transfected into MDA-MB-231 cells with FuGene®6 Transfection Reagent (Roche Diagnostics) at 37°C for 24 h, according to the manufacturer's instructions. Cells were incubated at 37°C with 2.2 µg/ml puromycin (Sigma-Akdrech, Merck KGaA) for 2 weeks. puromycin-resistant colonies (~20) were obtained and cultured with the medium containing puromycin (2.2 µg/ml) in 100 mm-diameter dishes. When the cell confluency reached 80%, dishes were provided to carry out western blot analysis and to gain FKD MDA-MB-231 cells, respectively. Transfection of the pLKO.1-puro plasmid vector without shRNA against fascin was used to generate non-FKD MDA-MB-231 cells.
8
Lentiviral Overexpression of KLK8 in Pancreatic Cancer Cells
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The human pancreatic cancer cell line Mia-paca-2, Panc-1 and human embryonic kidney cell line 293T (293T) were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Mia-paca-2 and Panc-1 cells were cultured in DMEM (Invitrogen). All medium was supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Invitrogen). KLK8 overexpression was performed using a lentiviral packaging system. To construct overexpressing exogenous KLK8 cell lines, full-length KLK8 (NM_144505) was cloned into the expression vector Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin (Shanghai Genechem Co. Shanghai, China) and transfected into Mia-paca-2 and Panc-1 cell lines according to the manufacturer’s instructions. Briefly, Mia-paca-2 and Panc-1 cells were placed in 6-well plates at a density of 1 × 10^5cells/well the day before infection. The next day lentivirus were added in cell culture medium. Viruses were removed 24 h after infection and fresh cell culture medium was added. 72h after transfection, puromycin (2 µg/ml; Roche, USA) was added into the cell culture medium to generate stable KLK8-overexpression cell line four weeks later. Antibiotic-resistant cells were pooled for subsequent analysis.
9
Modulating miR-410 Expression in Lung Cancer
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MiR-410 overexpression, inhibition and scrambled control lentivirus solutions were purchased from GenePharma (Shanghai GenePharma Co. Ltd, Shanghai, China). MiR-410 and its interfered siRNA sequences were as follows: miR-410, AAUAUAAC ACAGAUGGCCUGU; miR-410-siRNA, UUAUAUUGU GUCUACCGGACA. Briefly, A549 or H1299 cells cultured in 24-well plates were infected with lentivirus particles or scrambled control clone with Polybrene (5 μg/ml; Sigma, St.Louis, MO, USA). Medium containing lentivirus particles was replaced with fresh medium 24 h post-infection. Stable cells were selected after 72 h infection using puromycin (2 μg/ml; Roche, USA) by 3–4 weeks. The stable cells lines were further identified by detecting miR-410 expression by qRT-PCR. Antibiotic-resistant cells were pooled for subsequent analysis.
10
Overexpression and Knockdown of ZHX2 in Cancer Cells
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ZHX2 overexpression and its control lentivirus were obtained from HanBio (Shanghai, China). AGS cells cultured in six‐well plates were infected with overexpressing lentivirus particles or scrambled control clone with polybrene (5 µg·mL−1; Sigma, St Louis, MO, USA). Stable cells overexpressing ZHX2 were selected using puromycin (Roche, Indianapolis, IN, USA) after transfection. The small interfering RNA used to interfere with the expression of ZHX2 and the respective negative control were synthesized by Ribio (Shanghai, China) and BGC‐823 cells were transfected with small interfering RNA using Lipo2000 (Invitrogen, Carlsbad, CA, USA).
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