*4.2. Proliferation Assay*

Human MNNG/HOS osteosarcoma cell line, A375 melanoma, A549 lung cancer, U251 glioblastoma, MDA231 breast cancer and Caco2 colon cancer cell lines were purchased from the American Tissue Cell Collection (ATTC, Molsheim, France). Human MNNG/HOS osteosarcoma cell line (ATCC) was cultured with DMEM high glucose, pyruvate, nonglutamine from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 10% of fetal bovine serum (FBS, Thermo-Fisher). Human A375 melanoma cell line (ATCC) was cultured with DMEM 4.5 g/L high glucose, pyruvate, non-glutamine from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). Human A549 lung cancer cell line (ATCC) was cultured with DEMEM/F12 (Sigma Aldrich), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). Human U251 glioblastoma cell line (ATCC) was cultured with DMEM 4.5 high glucose, pyruvate, non-glutamine from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). Human MDA231 breast cancer cell line (ATCC) was cultured with L-15 media from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). Human Caco2 colon cancer cell line (ATCC) was cultured with DMEM 4.5 g/L high glucose, pyruvate, non-glutamine from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher).

Cells were incubated at 37 ◦C with humidity saturated controlled atmosphere and 5% CO2. At confluence, cells are detached using Trypsin (Thermo-Fisher) and washed to neutralize the enzyme. MNNG/HOS cells were seeded in triplicate at 5000 cells per well (50 μL) with 50 μL of culture medium (to measure the background) for 4 h, the time necessary for cell seeding, in an E-Plate view 96 (Chem Agilent, Santa Clara, CA, USA) before adding 100 μL of compound at the three concentrations (50, 100 and 200 μg mL−<sup>1</sup> for MNNG/HOS cells, 100 μg mL−<sup>1</sup> for A375, A549, U251 cells). The choice of these concentrations provided a wide range to study a possible dose dependent response. Proliferation curves are normalized respect the time point of drug incorporation. The plate was

monitored for 160 h (MNNG/HOS cells), 140 h (Caco2 cells) and 95 h (A375, A549, U251, MDA231 cells) using a RTCA instruments (Agilent and ACEA, Santa Clara, CA, USA). Experiments have been done in triplicates and repeated twice. Statistical tests have been conducted using Regression Data Analysis Tool in Excel®.

## *4.3. Viability Assay*

MNNG/HOS cells were seeded in triplicate at 3000 cells par well (50 μL) with 50 μL of culture medium for 4 h in an E-Plate view 96 (Chem Agilent) before adding 100 μL of compound at the three concentrations (50, 100 and 200 μg mL−1). The choice of these concentrations provided a wide range to study a possible dose dependent response. The plate was left 160 h: the volume of each well was reduced to 100 μL before adding 10 μL of 5 mg/mL MTT (Sigma-Aldrich) and incubating for at least 3 h at 37 ◦C and 5% CO2. After this time, the liquid was removed and 200 μL of DMSO was added to each well to dissolve the formed formazan crystals before proceeding to the colorimetric quantification with a multi-well spectrophotometer (Victor 3x from PerkinElmer, Villebon-sur-Yvette, France) at the wavelength of 500–600 nm.
