Cal51

TNBC breast cancer cell lines, including MDA-MB-231 and Cal-51, were obtained from the ATCC, Middlesex, UK. All cell lines were cultured in DMEM, complemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), and 1% penicillin–streptomycin. Before the experiment, the cell lines were tested for the presence of mycoplasma with MycoAlert (Lonza, Verviers, Belgium).
For lentiviral transduction, HEK293T cells were transfected with 3rd generation packaging plasmids including pVSVG, pRSV-Rev and pMDL g/p RRE (kindly provided by Didier Trone through Addgene, Cambridge, MA, USA) and shERWOOD UltramiR Lentiviral Inducible shRNA target gene set for gene HMGA2 or non-targeting shRNA (TransOMIC Technologies, Huntsville, AL, USA), using Lentifectin according to the manufacturer’s recommendations. shRNAs identifiers were: HMGA2-shRNA1 (ULTRA-3398612), HMGA2-shRNA2(ULTRA-3398613), and HMGA2-shRNA3 (ULTRA-3457905). After 72 h, media containing lentivirus was collected, filtered and stored at -80 degrees.
TNBC cells were seeded at a density of 20,000 cells/cm². The next day, the cells were transduced by adding media containing lentivirus particles and 5 μg/mL of polybrene for 24 h, after which the lentiviral-positive cells were selected by addition of 0.5 μg/mL of puromycin.

HCC1937, CAL-51, MDA-MB-231, and MDA-MB-468 were purchased from ATCC and maintained in DMEM (Corning) containing 10% FBS (Atlas Biologicals, Ft. Collins, CO, USA) with 1% Pen/Strep and 1% non-essential amino acids (Corning, Tewksbury, MA, USA). Cells were routinely tested for mycoplasma and were authenticated at the Barbara Davis Center for Diabetes Molecular Biology Service Center (Aurora, CO, USA).

All BC and adjacent noncancerous breast tissues in this study were collected from Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital). The tissues were immediately snap frozen in liquid nitrogen until RNA extraction. Patients did not receive chemotherapy or radiotherapy and signed informed consent. This study was approved by the Medical Ethics Committee of Cancer Hospital of the University of Chinese Academy of Sciences. The BC cell lines (CAL51, BT549, MCF7, and MDA-MB231) and the human mammary epithelial cell line (MCF-10A) were purchased from ATCC (Manassas, VA, USA) and cultured according to the manufacturer’s instructions.

HeLa, BT-549, CAL-51, H460, HCT-116, PSN-1, MRC5 and HFL-1 cells were purchased from American Type Culture Collection (ATCC). SQ20B cells were kindly provided by Dr. Ralph Weichselbaum (University of Chicago). HeLa, SQ20B, BT-549, HCT-116 and PSN-1 were maintained in Dulbecco's modified Eagle's medium (DMEM), MRC5 in MEM and HFL-1 in F-12 Hams media. All media (Sigma) were supplemented with 10% FBS. All cell lines were tested for mycoplasma using MycoAlert (Lonza). Cell lines were never grown beyond four months after purchase. The HeLa cell line, which had been cultured for a longer period, was authenticated by LGC standards (ATCC) by short tandem repeat (STR) profiling. The SQ20B cell line was authenticated by DNA sequencing methods and has not varied over time.

The Research Resource Identifiers (RRIDs) for all cell lines used are provided in Table S2. HMEC (human mammary epithelial cells) were a gift from M. R. Stampfer [39 ]. All other cell lines were purchased from DSMZ (“Deutsche Sammlung von Mikro-Organismen und Zellkulturen,” Braunschweig, Germany): CAL-51, HCC1143, HCC1937, and KPL-1, or ATCC (American Type Culture Collection, Manassas, USA): AU565, BT-474, CAMA-1, Hs 578T, Hs 578Bst, MCF-7, MCF-10A, MCF-10F, MDA-MB-231, MDA-MB-453, MDA-MB-468, SK-BR-3, T-47D, and ZR-75-1. Cell culture conditions of all cell lines were described previously [40 (link)]. DSMZ and ATCC authenticate all cell lines by STR profiling before distribution. Genomic DNA and total RNA were isolated from all cell lines immediately after receipt, i.e., within three to eight passages [40 (link),41 (link)].