Hbl 100

Human embryonic kidney cells (HEK293T) were obtained from Riken Cell Bank (Ibaraki, Japan). Human cancer cell lines U373MG (astrocytoma), H1299 (non‐small‐cell lung cancer), HCT116 (colorectal adenocarcinoma), and HBL100 (breast carcinoma) cells were purchased from ATCC (Rockville, MD, USA). Human mammary epithelial cells MCF10A p53^+/+ or MCF10A p53^−/− were purchased from Sigma Aldrich (St. Louis, MO, USA). HBC4 (breast carcinoma) cells were gifted from Dr. T. Yamori (Japanese Foundation for Cancer Research, Tokyo, Japan). HCT116 p53^+/+ and HCT116 p53^−/− cell lines were gifts from Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA). HEK293T and U373MG cells were transfected with plasmids using Fugene6 (Promega, Madison, WI, USA), and Lipofectamin LTX (Invitrogen, Carlsbad, CA, USA), respectively. Small interfering RNA oligonucleotides, commercially synthesized by Sigma Genosys (Woollands, TX, USA), were transfected with Lipofectamine RNAiMAX reagent (Invitrogen). Sequences of siRNA oligonucleotides are shown in Table S1.

Breast cancer cell lines MCF-7, MDA-MB-231, BT-549 and HBL-100 (all obtained from ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum (FCS) and 100 IU ml⁻¹ penicillin/streptomycin. These cell lines correspond to different molecular subtypes of breast cancer, with hormone-dependent MCF-7 expressing ERs (ER+) and MDA-MB-231, BT-549 and HBL-100 being characterised as triple-negative cell lines, lacking in receptors for oestrogen, progesterone and human epidermal growth factor (ER− PR− HER2−) and, hence, hormone-independent. The LCC1, LCC2 and LCC9 (hormone-independent cells established by derivation of selected subpopulations of MCF-7 cells (Brünner et al, 1993b (link), 1997 (link); Thompson et al, 1993 (link))) were cultured in phenol red-free DMEM supplemented with 5% heat-inactivated FCS charcoal-stripped of steroids. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were grown to confluence with periodic medium changes and collected by brief incubation with trypsin/ethylenediaminetetraacetic acid solution. All cell lines were authenticated by short tandem repeat profiling undertaken by the Public Health England (Salisbury, UK) in December 2014.