*2.2. Cell Culture and Treatments*

All cell lines were from ATCC. HCC1569 (ER<sup>−</sup>, Pr<sup>−</sup>, Her2 +), HCC1954 (ER<sup>−</sup>, Pr<sup>−</sup>, Her2 +) and BT-474 cells (ER +, Pr+/<sup>−</sup>, /Her2 +) were maintained in RPMI-1640 or Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine, antibiotics and 10% heat-inactivated foetal bovine serum (FBS) all from Corning (New York, NY, USA), according to ATCC indications. MCF10A cells were maintained in Dulbecco's modified Eagle's medium DMEM/F-12 medium containing 5% horse serum (Thermo Fisher Scientific, Waltham, MA, USA) hydrocortisone (0.5 μg/mL) (Sigma-Aldrich), insulin (10 μg/mL) (Sigma-Aldrich), Epidermal growth factor, EGF (20 ng/mL) (Sigma-Aldrich), cholera toxin (100 ng/mL) (Sigma-Aldrich), penicillin (100 units/mL) and streptomycin (100 μlg/mL) (Corning).

Cells (at 5000 cells/cm<sup>2</sup> or otherwise indicated) were cultured in a humidified incubator in an atmosphere of 5% CO2 at 37 ◦C. Before any experiment, cells were detached by mild trypsinization, washed, plated in medium containing 10% FBS, and allowed to recover for 24 h. Cells were treated with DHA dissolved in DMSO (Sigma-Aldrich) or with T-DM1. Control media contained the same amount of DMSO-vehicle (<0.1%). All cell lines were tested for mycoplasma contamination regularly using MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and were authenticated by STR sequencing (BMR Genomics, Padoa, Italy).
