*2.17. Cell Transfection*

Retroviruses were produced by transfection of Phoenix-Ampho packaging cells with pBABE-puro, AA-TCTP-pBABE, and WT-TCTP-pBABE using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, supernatants containing the retroviral particles were collected and frozen at −80 ◦C until use. MCF10A cells were infected with diluted supernatant in the presence of 8 μg/mL Polybrene (Sigma-Aldrich) overnight, and cells containing the pBABE, AA-TCTP-pBABE, and WT-TCTPpBABE constructs were selected with puromycin (1 μg/mL) (Sigma-Aldrich) 48 h after infection. After 10 days in selective medium, the three pools referred to empty vector (MCF10A-pBABE), the wild type TCTP protein (WT-TCTP), the Ser46Ala Ser64Ala double mutant TCTP (AA-TCTP), were isolated. The puromycin selective pressure was removed 24 h before experimental procedures.

### *2.18. Evaluation of Cell Sensitivity to Combined Treatment*

Cells were plated in triplicate in 96-well and treated with DHA, T-DM1, and with the DHA/T-DM1 combination. Growth inhibition was calculated as the percentage of viable cells compared to untreated cells by the CellTiter-Glo Luminescent Cell Viability assay (Promega, Madison, WI, USA) The CompuSyn software program has been used to calculated synergistic, additive or antagonistic effects. This program is based on the Median-Effect Principle (Chou) and the Combination Index–Isobologram Theorem (Chou-Talalay) [45]. Because all terms in the equations are ratios, all the dose units become dimensionless quantities. Drug can be different units. The combination index (CI) indicates a quantitative measure of the degree of drug interaction in terms of synergistic (CI < 1), additive (CI = 1) or antagonistic effect (CI > 1). DRI is the dose-reduction index and it is a measure of how many-fold the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses of each drug alone.
