The following cell lines were used in this study: human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs; male)53 (link), HeLa (American Type Culture Collection (ATCC) CCL-2), HEK293T (ATCC CRL-11268), T-REx-293 (Life Technologies), BS-C-1 (ATCC CCL-26) and RK13 (ATCC CCL-37). All cell lines were cultured in DMEM (Gibco), supplemented with 10% FBS (PAN Biotech) and 50 μg ml−1 penicillin–streptomycin (Gibco). T-REx-293-derived cells that were stably transfected with pcDNA4/TO plasmids were further supplemented with 10 μg ml−1 blasticidin (Thermo Fisher) and 100 μg ml−1 zeocin (Gibco). HEK293T cells that were transduced to overexpress Myc-tagged proteins10 (link) were supplemented with 1 μg ml−1 puromycin (Invivogen). A complete list of cell lines is described in Supplementary Table 1 .
T rex 293
Manufactured by Thermo Fisher Scientific
Sourced in United States
The T-REx-293 is a genetically engineered human embryonic kidney cell line that enables the inducible expression of recombinant proteins. It contains a tetracycline-regulated expression system that allows for tight control over the expression of target genes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
Hek293t, by American Type Culture Collection (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Dmem high glucose, by Merck Group (4 mentions)
Polyfect, by Qiagen (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
T rex 293 cells, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Merck Group (2 mentions)
Blasticidin, by Thermo Fisher Scientific (2 mentions)
Bs c 1, by American Type Culture Collection (2 mentions)
Zeocin, by Thermo Fisher Scientific (2 mentions)
Hek293t, by InvivoGen (2 mentions)
Fetal bovine serum (fbs), by PAN Biotech (2 mentions)
Puromycin, by InvivoGen (2 mentions)
Leibovitz s l 15 media, by Corning (2 mentions)
Bhk 21, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Omega Scientific (2 mentions)
Dubelco s modified eagle s medium, by Corning (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
26 protocols using t rex 293
1
Cell Line Cultivation Protocol
2
HEK293T and 293 T-REx Cell Culture
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HEK293T (ATCC #CRL-1573) and 293 T-REx (Thermo Fisher Scientific #R78007) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum and antibiotic–antimycotic. 293 T-REx stable cell lines were generated by recombinase-mediated DNA recombination using the Flp-In system (Thermo Fisher Scientific) and maintained in DMEM containing 100 µg/ml hygromycin B. Recombinant protein expression was induced by treating 293 T-REx cells with 1 µg/ml tetracycline for 24 h. Transfections were performed using PolyJet (SignaGen) or PolyEZ (MoCellutions) reagents.
3
Construction and Characterization of SOD1 Minigene
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Construction of the SOD1 minigene, pcDNA_SOD1, and generation of stable minigene cell lines was described previously [20] (link). A splice-defective mutant, pcSOD187M/TO, in which the native U1 consensus sequence, UG GU AAGU was converted to UG GUUGGG to prevent binding of U1 snRNP at the 5′ splice site, was generated by site directed mutagenesis using a QuikChange Lightning SDM Kit (Agilent Technologies) with primers W187F 5′-CATCATTGGCCGCACACTGGTGGTTGGGTTTCATAAAAGGATATGCATAAAAC-3′ and W187R 5′-GTTTTATGCATATCCTTTTATGAAACCCAACCACCAGTGTGCGGCCAATGATG-3 according to the manufacturer’s protocol.
T-REx-293 and T-REx-HeLa cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% fetal calf serum, 0.1 µg/ml streptomycin, 100 units/ml penicillin, and 5 µg/ml blasticidin. Plasmids pcSOD1/TO and pcSOD187M/TO were transfected into T-REx-293 cells using Effectene transfection reagent according to the manufacturer’s protocol (Qiagen). Cells in which the minigene was stably integrated were selected in DMEM media containing 250 µg/ml zeocin. Zeocin-resistant colonies were expanded then tested for induction of expression by tetracycline (TET) using qRT/PCR. Cell lines overexpressing E. coli RNase H were generated as described previously [20] (link).
T-REx-293 and T-REx-HeLa cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% fetal calf serum, 0.1 µg/ml streptomycin, 100 units/ml penicillin, and 5 µg/ml blasticidin. Plasmids pcSOD1/TO and pcSOD187M/TO were transfected into T-REx-293 cells using Effectene transfection reagent according to the manufacturer’s protocol (Qiagen). Cells in which the minigene was stably integrated were selected in DMEM media containing 250 µg/ml zeocin. Zeocin-resistant colonies were expanded then tested for induction of expression by tetracycline (TET) using qRT/PCR. Cell lines overexpressing E. coli RNase H were generated as described previously [20] (link).
4
Cell Culture Maintenance Protocol
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293T cells, T-REx 293 (Invitrogen) cells, and JSQ3 cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin (P/S). CEM-GFP, HCC1569, and OVCAR5 cells were maintained in RPMI medium with 10% FBS and 0.5% P/S. The CEM-GFP HIV reporter cell line was obtained from the NIH AIDS Reagent Program [53 (link)]; the breast cancer cell line HCC1569 from ATCC; the head and neck cancer cell line JSQ3 from Dr. Mark Herzberg (University of Minnesota); and the ovarian cancer cell line OVCAR5 from the Mayo Clinic ovarian cell line repository.
5
Cell Culture Methods for Stable Lines
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RPE hTERT and ARPE-19 cells were grown in DMEM F12 media with 10% FBS (Sigma-Aldrich), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine. mIMCD-K2 cells were a gift from B. Stanton (Dartmouth College, Hanover, NH). T-Rex-293 (Invitrogen), IMCD3 Flp-In, and Phoenix A (PhA) cells (Indiana University National Gene Vector Biorepository) were cultured in DMEM high glucose (Sigma-Aldrich; supplemented with 10% FBS, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). Transfection of plasmids was done with Polyfect (QIAGEN). Stable cell lines were generated by retroviral infection or transfection. NIH 3T3 Flp-In cell lines stably expressing Gpr161WT/V158E-LAP (Gpr161 followed by Speptide-PrecissionS-EGFP [LAP5]) were generated by retroviral infection and antibiotic selection (Pal et al., 2016 (link)). IMCD3 Flp-In cells stably and inducibly expressing 6×Myc-tagged TULP3 N terminus (1–183 aa) were generated by lentiviral infection of the pLVX TetOne vector (Takara Bio Inc.) with the insert. In many cases, stable lines were flow sorted and further selected for GFP.
6
Cell Culture Protocols for Multiple Cell Lines
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T-Rex-293 (Invitrogen), IMCD3 Flp-In (IMCD3; American Type Culture Collection; gift of Peter Jackson), Phoenix A (PhA; Indiana University National Gene Vector Biorepository), and 293FT cells were cultured in DMEM high glucose (Sigma-Aldrich; supplemented with 10% cosmic serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). NIH 3T3 Flp-In (Thermo Fisher) cells were cultured in DMEM high glucose media (D5796; Sigma) with 10% Bovine calf serum (BCS; Sigma-Aldrich), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine. 3T3-L1 cells (gift of Peter Michaely, UTSW) were cultured in DMEM high glucose (Sigma-Aldrich) media with 10% fetal bovine serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 4.5 mM glutamine, and 8 ng/ml biotin. Cell lines were tested negative for Mycoplasma using the Mycoplasma PCR Detection Kit (Genlantis). Transfection of plasmids was done with Polyfect (QIAGEN) or polyethylenimine (PEI) max. Stable cell lines were generated by retroviral infection or transfection. In many cases, stable lines were flow sorted and further selected for GFP. Control and Tulp3 ko MEFs were from embryonic day 13.5 embryos (Legue and Liem, 2019 (link)). Immortalized WT and Arl13b mutant MEFs were gifts from Tamara Caspary (Larkins et al., 2011 (link); Gigante et al., 2020 (link)).
7
Cell Culture Protocol for TREx-293 and VCaP
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TREx-293 and VCaP cell lines were obtained from Invitrogen and ATCC, respectively. Cells were routinely grown in T-75 cm2 flasks in Dulbecco's Modified Eagle Medium (DMEM) high glucose (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Sigma).
8
Generation of Inducible NleL-expressing T-REx 293 Cell Lines
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To create T-REx 293 Ha-NleL cell lines, pcDNA4/TO/N-2xHA containing full-length, codon-optimized NleL was linearized using ScaI and transfected into T-REx 293 cells (Invitrogen). Cells were selected with 150 μg/mL zeocin and individual colonies were expanded and screened for NleL expression by western blotting with an HA-antibody. NleL expression was induced with 1 μg/mL doxycycline for 12 hr at 70% confluency and TUBE pull-downs were performed as described below.
9
Influenza A/WSN/33 (H1N1) Virus Propagation
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The influenza A/WSN/33 (H1N1) virus (WSN) used in this study was propagated in Madin–Darby canine kidney (MDCK) cells, which were grown in Dulbecco's modified Eagle's medium (DMEM; Gibico) containing 10% fetal bovine serum (FBS). Viral titration was by plaque-formation assays on MDCK cells. The human lung epithelial carcinoma cell line A549 was maintained in F-12 medium (Invitrogen) supplemented with 10% FBS. Human embryonic kidney 293T (HEK293T) cells were grown in DMEM (Sigma) containing 10% FBS. The tetracycline-regulated HEK293 cell line (T-REx-293; Invitrogen) expressing HA-tagged ZFP36L1 was established as described (36 (link)) and grown in DMEM (Sigma) containing 10% tetracycline-free FBS plus 5 μg/ml blasticidin (InvivoGen) and 250 μg/ml hygromycin (InvivoGen). Doxycycline (Dox) was from Clontech. MG132 was from Merck. Polyinosine-polycytidylic acid (Poly[IC]) low molecular weight was from InvivoGen. Rabbit and mouse monoclonal anti-HA antibodies, rabbit anti-BRF1/2 (ZFP36L1) and rabbit anti-CNOT6 were from Cell Signaling. Rabbit anti-exosome C5 antibody was from Abcam. Rabbit anti-XRN1 antibody was from Novus Biologicals. Rabbit polyclonal anti-influenza A H1N1 antibodies were from Genetex. Rabbit polyclonal anti-IRF-3 antibody was from Santa Cruz Biotechnology.
10
Culturing Cell Lines for GPR161 Studies
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T-REx-293 (Invitrogen), IMCD3 Flp-In, and Phoenix A (PhA) cells (National Gene Vector Biorepository) were cultured in DMEM high glucose (Sigma-Aldrich; supplemented with 10% FBS [Sigma-Aldrich], 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). NIH 3T3 Flp-In cells (Invitrogen) were grown in DMEM high glucose (supplemented with 10% bovine calf serum [Sigma-Aldrich], 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine). NIH 3T3 Flp-In cell lines stably expressing wild type/mutant Gpr161GFP (Gpr161 followed by Speptide-PrecissionS-EGFP [LAP5]), and untagged receptor constructs were generated using transfection or retroviral infection and antibiotic selection (Mukhopadhyay et al., 2013 (link)). ARPE-19 cells were grown in DMEM F12 (Sigma-Aldrich) media with 10% FBS (Sigma-Aldrich), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 4.5 mM glutamine. Stable CAMYEL T-REx-293 cells (Invitrogen) inducibly coexpressing untagged mouse Gpr161 were generated by transfection of plasmids and antibiotic selection. Transfection of plasmids was done with either Polyfect (QIAGEN) or Bio-T (BioLand Scientific).
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