Cama 1

MCF7, ZR75, CAMA-1 (ATCC) and SUM52 (Asterand) and non-LTED eveR derivatives were cultured in RPMI (Gibco) with 10% FBS (Sigma), 1% L-glutamine and antibiotics. For cells purchased from ATCC, cell lines were validated using short tandem repeat (STR) profiling and data was compared to the ATCC database. All LTED and LTED-EveR lines were generated and cultured in phenol red-free media + 10% charcoal dextran-treated fetal bovine serum (Hyclone), 1% L-glutamine and antibiotics. Everolimus resistance was generated by subjecting the parental or LTED derivative of each cell line to increasing concentrations of everolimus up to 500nM and maintained at this concentration. Generation of everolimus resistant pools took four to nine months depending on the cell line. During assays, cells were washed out of everolimus during plating and treated the following day with indicated compounds. MYC siRNA experiments were performed using either siRNA #1 (Dharmacon, D-003282-14-0020) or siRNA#2 (LifeTech, #S9130) or a non-targeting siRNA control (Dharmacon). Lines were transfected at a final concentration of 5nM and the following day cells were plated for growth assays. pTRIPZ constructs were obtained from Open Biosystems and viral transduction was performed as per manufacturer's instructions with packaging plasmids from Open Biosystems.

The human breast cancer cell lines MCF-7, T47D, MDA-MB-231 and MDA-MB-468 were purchased from ATCC and were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) (Biosera, Boussens, France), 1 % sodium pyruvate, 1 % HEPES and penicillin/streptomycin (100 U/ml and 100 μg/ml respectively); CAMA-1 (also purchased from ATCC) was cultured in MEM/EBSS supplemented with 10 % FBS and penicillin/streptomycin, and SUM-149 and SUM-159 were cultured in F-12 HAM’S medium supplemented with 5 % FBS, 1 mM L-Glutamine, 1 μg/ml hydrocortisone (BD BioScience, San Diego, CA, USA) and 5 μg/ml insulin (Novo Nordisk A/S, Måløv, Denmark). The SUM-149 and SUM-159 cell lines were produced by Professor S Ethier. Media and supplements were purchased from Thermo Scientific HyClone (South Logan, UT, USA) unless otherwise stated.

CAMA1, MAD-MB-361, and T47D cells were purchased from ATCC in 2019 and grown in DMEM supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution. Long-term estrogen-deprived (LTED) cells were generated and maintained, as described in the previous report (Miller et al. 2010 (link)).