Rad001

In this study, we used A375 human melanoma cell lines, obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). In some experiments we used also the human melanoma cell lines WM266-4 (from ATCC) and M21 (kindly provided by Dr. Antony Montgomery, The Scripps Research Institute, La Jolla, CA, USA). Melanoma cells were cultivated in Dulbecco’s Modified Eagle Medium high glucose (DMEM 4500, EuroClone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Boerhinger Mannheim, Binger Strasse, Ingelheim am Rhein, Germany), at 37 °C in a humidified atmosphere containing 90% air and 10% CO₂. Viability of the cells was determined using a trypan blue exclusion test. Cultures were periodically monitored for mycoplasma contamination using Chen’s fluorochrome test [26 (link)].
A375 melanoma cells resistant to PLX4032 were kindly provided by Laura Poliseno from University of Pisa and they were obtained as explained in Reference [27 (link)]. PLX4032-resistant A375 melanoma cells were maintained without PLX4032 overnight before the start of the experiment.
According to the experiments, cells were treated with Oleuropein glucoside (purity ≥ 90%) (Extrasynthese S.A., Lyon, Nord-Genay, France), DTIC (Sigma Aldrich, Milan, Italy), RAD001 (MedChem Express, Stockholm, Sweden) or PLX4032 (MedChem Express, Stockholm, Sweden).

The migration assay was performed using 8.0 μm pore size, fibronectin-coated inserts in 24-well plates (Corning-Falcon cat#353097). Triplicates of 3 × 10⁴ MM27 shLuc and ShcD-silenced cells were plated in the upper chamber in serum-free medium. Vehicle (DMSO) or Dabrafenib 2.5 nM (GSK2118436A, Active Biochem, Cat#A-1220), Trametinib 0.5 nM (GSK-1120212, Active Biochem, Cat#A-1258), and Everolimus 2.5 μM (RAD001, MedChemExpress, Cat#HY-10218) were added alone or in combination (Dabrafenib/Trametinib and Dabrafenib/Everolimus, same doses as single testing) to the medium. The complete medium was added to the lower chamber. After 48 h, cells that migrated to the lower surface of the inserts were stained with 0.5% Crystal Violet. Four images of each insert were acquired and analyzed with the ImageJ Software.

The following antibodies for the purpose of Western blotting were acquired from Cell Signaling Technologies: pAKT S473 (Catalog number 4060), PAN-AKT (#2920), Phospho-p44/42 MAPK (#4370), p44/42 MAPK (#4695), S6 Ribosomal Protein (#2317), Phospho-S6 Ribosomal Protein (#4858), Phospho-p70S6 Kinase Thr389 (#97596), p70S6 Kinase (#9202), Phospho-4EBP1 Thr37/46 (#2855), 4EBP1 (#9452). Antibodies against Beta-Actin were purchased from GeneTex (catalog number 109639). MAPK inhibitors were purchased from Tocris: FR180204 (catalog number 3706), U0126 (catalog number 1144), PD980595 (catalog number 1213). Sapanisertib (Cat# HY-13328) and Rapalink-1 (Cat# HY-111373) were purchased from MedChemExpress. Torin-1 (Cat #4247) was purchased from Tocris. PI3K/AKT/mTOR inhibitors BKM120 (Buparlisib; Novartis), AZD5363 (Capivasertib; Selleckchem), and RAD001 (Everolimus, MedChemExpress Cat# 159351–69–6) were generously provided by Dr Kathleen Millen.