MCF7 and T47D cells with a doxycycline (DOX)-inducible Y537S or D538G (DOX-Y537S or DOX-D538G) mutation in ER (6 (link)), were grown in DMEM and RPMI, respectively [supplemented with 10% FBS, 10 μg/mL penicillin-streptomycin-glutamine (PSG), and 500 μg/mL Geneticin], hereafter referred to as full media (FM) conditions. DOX-inducible ER-mutant MCF7 and T47D cells (MCF7 DOX-Y537S, MCF7 DOX-D538G, and T47D DOX-Y537S) were treated with 1 μg/mL DOX in FM for 3 days to induce the expression of the mutation and subsequently maintained in phenol-red-free DMEM or RPMI supplemented with 10% charcoal-stripped FBS, PSG, and Geneticin, hereafter referred to as HD conditions. MCF7 cells with a stable TALEN knock-in Y537S (KI-Y537S) or D538G (KI-D538G) ER mutation (6 (link)) were grown in DMEM supplemented with 10% FBS and PSG. Palbociclib-sensitive (PalboS) and palbociclib-resistant (PalboR) T47D cells (23 (link)) and MCF7 cells (24 (link)) were grown in DMEM supplemented with 10% FBS and PSG, with or without palbociclib 100 nmol/L for the PalboS cells, and with palbociclib 1 μmol/L for the PalboR cells. Cells were authenticated by short tandem repeat profiling (Bio-Synthesis) and regularly tested for Mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza) according to the manufacturer's instructions.
Protocol detail
Engineered ER-mutant Breast Cancer Cell Lines
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Corresponding Organization :
Other organizations : Harvard University, Dana-Farber Cancer Institute, Garvan Institute of Medical Research, Massachusetts Institute of Technology, Baylor College of Medicine, University of Naples Federico II, Hospital of Prato, Imaging Center
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Variable analysis
independent variables
- Mutation in ER (Y537S or D538G)
- Doxycycline (DOX) induction
- Palbociclib treatment (100 nmol/L for PalboS cells, 1 μmol/L for PalboR cells)
- Cell growth and proliferation
- Cell lines (MCF7 and T47D)
- Cell culture media (DMEM and RPMI)
- Supplementation (10% FBS, 10 μg/mL penicillin-streptomycin-glutamine, 500 μg/mL Geneticin)
- Charcoal-stripped FBS for hormone-depleted (HD) conditions
- Cell authentication and Mycoplasma testing
- Positive control: DOX-inducible ER-mutant MCF7 and T47D cells
- Negative control: MCF7 cells with stable TALEN knock-in Y537S or D538G ER mutation
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