All cell lines were maintained in minimum essential medium (MEM) (Gibco, 11095080) supplemented with 10% fetal bovine serum (FBS) (HyClone, SH30070.03) and 1% penicillin and streptomycin (Thermo Fischer, 15140122). MCF7, T47D, and BT474 parental cell lines were obtained from ATCC. All cell lines were authenticated and negative for mycoplasma contamination. When indicated, cells were cultivated in an ultralow adherence culture dish (Corning, 3262). MCF7 cell lines constitutively expressing SOD2 mutants were obtained by lentiviral transduction and selected using appropriate antibiotics. SOD2 mutants were cloned into lentiviral vector pLenti-CMV-MCS-GFP-SV-puro (29 (link)) (Addgene, 73582), by using an existing SOD2 sequence as previously described (13 (link)) with the addition of nuclear localization signal (NLS), nuclear exclusion signal (NES), or Flag-tag. HEK-293T cells were transfected using Lipofectamine 3000 (Invitrogen, L3000001) together with lentiviral packaging constructs 8.91 and VSVG. Stably transduced MCF7 cells were selected using 1 µM/mL puromycin (Gibco, A1113803).
Protocol detail
Generating SOD2 Mutant MCF7 Cell Lines
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293t cells
Antibiotics
Cell lines
Cells
Dish
Fetal bovine serum
Lipofectamine
Mcf7 cells
Mycoplasma
Nuclear localization signal
Parental
Penicillin
Puromycin
Sod2
Streptomycin
Vector
Corresponding Organization :
Other organizations : Northwestern University, Medical College of Wisconsin, Roosevelt University, Wake Forest University
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Variable analysis
independent variables
- SOD2 mutants (SOD2 with nuclear localization signal (NLS), nuclear exclusion signal (NES), or Flag-tag)
- Cell line characteristics/phenotypes (not explicitly mentioned)
- Cell culture medium (MEM supplemented with 10% FBS and 1% penicillin and streptomycin)
- Parental cell lines (MCF7, T47D, and BT474)
- Cell line authentication and mycoplasma testing
- Positive control: Parental cell lines (MCF7, T47D, and BT474)
- Negative control: Not explicitly mentioned
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