Mda mb 361

MCF10A (Cat#CRL-10317, RRID:CVCL_0598), MCF7 (Cat# HTB-22, RRID:CVCL_0031), BT474 (Cat# HTB-20, RRID:CVCL_0179), and MDA-MB-361 (Cat#HTB-27, CVCL_0620) cell lines were procured from ATCC (Manassas, VA). All cell lines used in this study were tested every two months and confirmed to be mycoplasma free using the MycoAlert^™ Mycoplasma Detection Kit (Lonza, Cat#LT07-218). MCF7 cells were grown in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS, 10 μg/ml human recombinant insulin, and antibiotics. MCF10A cells were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml human EGF, 500 ng/ml hydrocortisone, 10 μg/ml human recombinant insulin, 100 ng/ml Cholera toxin, and antibiotics. All other cell lines were cultured in DMEM supplemented with 10% FBS and antibiotics. All cell lines were maintained in an incubator set at 37°C and supplied with 5% CO₂ and less than passage 20.

T47D, MCF7, AU565, MDA-MB-231, MDA-MB-361, and ZR-75-1 cell lines were purchased from ATCC (American Type Culture Collection) in 2011. 4T1 cells were purchased from ATCC in 2013. SUM-159 were obtained from Dr. Jeffrey A. Frost (University of Texas Health Science Center at Houston), human mammary epithelial (HMLE) and HMLE ER-Twist cells were kindly provided by Dr. Sendurai A. Mani (University of Texas MD Anderson Cancer Center) in 2013. All cell lines used in this study were used at low passage and shown to be free of mycoplasma (MycoAlert Mycoplasma Detection Kit, Lonza #LT37-618). Except for SUM-159, all cell lines were obtained either from ATCC or the original source (HMLE). We did no further authentication on the SUM-159 cell line.
Additional procedures are described in Supplementary Materials and Methods.

Human primary epithelial cells (HMEC) were purchased from Invitrogen and human breast cancer MDA-MB-231, MDA-MB-157, BT549, MDA-MB-361, and T47D cells were purchased from ATCC (Rockville, MD). The cells were grown as recommended by the supplier with penicillin-streptomycin (Life Technologies Corporation, NY) at 37°C in 10% CO₂. HMLE and HMLE-Twist-ER cells were generous contributions from Professor R. Weinberg (The Whitehead Institute, Cambridge, MA) and were grown in Mammary Epithelial Growth Medium (MEGM) from Lonza with penicillin-streptomycin. 50 nM 4-hydroxytamixofen (Sigma) was added to the HMLE-Twist-ER cells to induce Twist-ER expression. HMLE-Twist-ER cells were treated with DMSO or 100 nM Concanamycin-A (VWR International) or 10 μM MG132 (EMD Millipore) for six hours prior to harvesting of total cell lysates for western blotting. MDA-MB-231 and MDA-MB-157 cells were treated with 0.5 μM ARQ197 for 24 hrs to assess effect on c-Met phosphorylation.

The Hap1 cell line (8 (link)) was kindly provided by Dr. Thijn Brummelkamp, Netherlands Cancer Institute. The 293FT cell line used to generate high-titer lentiviruses was obtained from ThermoFisher Scientific; the A549, PC3, MDA-MB-361, HCC-1954 and NCI-H1650 cell lines were purchased from ATCC, where they were validated by STR profiling, and used at passage numbers <5. The KOPN8 cell line was a generous gift from Professor Michael Cleary, Stanford University. The Phoenix-Ampho cell line used for retrovirus production was purchased from Allele Biotechnology (San Diego, CA).
Null alleles for genes were constructed using the CRISPR/Cas9 system. For Hap1 cells the oligos encoding the guide RNAs were cloned into pSpCas9(BB)-2A-GFP (PX458, Addgene Plasmid #48138 from Dr. Feng Zhang) (9 (link)). Single cells were sorted using flow cytometry, expanded and clones bearing null alleles were identified by Sanger sequencing and immunoblotting. For gene disruption in cancer cell lines, the gRNAs validated in the Hap1 cells were introduced into LentiCRISPR v2 (Addgene Plasmid #52961 from Dr. Feng Zhang) (10 (link)) for lentiviral-mediated delivery. The oligo sequences for guide RNAs are provided in the supplementary methods.

UACC812 cell line, which contains HER2 amplification, MDA-MB-361, and T47D cell lines, which harbor a PIK3CA active mutation (Aksamitiene et al., 2010 (link)), were purchased from ATCC. BT-474 cell line was a gift from Dr. Lewis Chodosh, MCF-HER2 and MCF-Neo cell lines were gifts from Dr. Mien-Chi Hung. To generate a cell line with conditional expression of c-Myc shRNA, shRNA oligonucleotides for c-Myc was cloned into pLKO-tet-on vector from Addgene. BT474 cells were transduced with either scramble or shRNA targeted against c-Myc mRNA. Cells were selected with puromycin. Wild-type c-Myc was cloned in MSCV-neo vector from Addgene. To obtain c-Myc impervious to shRNA knockdown site directed mutagenesis of this plasmid was performed to change 5’-CAGCAAC to 5’-ATCGAAT using a kit from Stratagene. All cell lines were cultured in DMEM with 10% FBS and 1% Penicillin/ Streptomycin and maintained at 37° C and 5% CO2.