Ly294002 ly

Migration of SH-SY5Y cells was carried out using the Ibidi Culture-Inserts assay (2 × 0.22 cm²; Ibidi, Regensburg, Germany) following the manufacturer’s instructions. Three hundred thousand cells were seeded on the culture-inserts. After 24 h, inserts were carefully removed and migration was analyzed using a BDS200 inverted microscope (Optec, Chongqing, China) at time 0 (insert removal) or following 24 and 48 h of EV treatment. To study the effects of AKT inhibition, cells were pre-treated with 10 μM of LY294002 (LY) (Cell Signaling Technology, Inc., Danvers, MA, USA) 1 h before EV treatment, and added every 24 h during medium refreshment. The migration rate was determined by analyzing the cell-free area with ImageJ Analysis Software 1.50i (National Institutes of Health, Bethesda, MD, USA).

Human thyroid follicular epithelial Nthy-ori 3-1 cells, obtained from European Collection of Authenticated Cell Cultures (ECACC 90011609) (Public Health England, Porton Down, Salisbury, UK) (Sigma Aldrich, St. Louis, MO, USA) were cultured at 37 °C in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin and 2 mM L-glutamine. Human thyroid cancer cell lines, TPC-1 (harboring RET-PTC rearrangement, BRAF WT/WT), characterized according to Schweppe et al., and kindly provided by A. Coppa (Department of Experimental Medicine, Sapienza University of Rome, Rome), were maintained in a 5% CO₂ culture humidified atmosphere, at 37 °C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS [29 (link)]. For experiments, cells were seeded and at sub-confluence (70%), were starved overnight using medium with 0.2% FBS, in order to reduce basal cellular activity [30 (link),31 (link)]. Then, the cells were stressed by H₂O₂ (Sigma-Aldrich, Milan, Italy) alone or combined with EGF (human recombinant, Sigma Aldrich) or MAPK and AKT inhibitors (PD98059 and LY294002, respectively). In particular, the cells were pretreated with 50 ng/mL EGF for 15 min or with PD98059 (PD) at 50 μM and LY294002 (LY) at 25 μM (Cell Signaling Technology, Beverly, MA, USA) for 1 h and then stressed by a supra-physiological H₂O₂ concentration added to the external medium.

The HCC cell lines HepG2, Hep3B, Huh7, PLC/PRF/5, HepG2-Luc(+) and Hep3B-Luc(+) were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), and 1% penicillin-streptomycin. The cells were incubated in a humidified atmosphere containing 5% CO₂ at 37°C. Where appropriate, cells were treated with the chemicals LY294002 (LY) (Cell Signaling Technology Inc., Danvers, MA) [33 (link)]; or Cns-A (Cytosporone A n-amyl ester, compound 10i) (Sigma-Aldrich, St. Louis, MO) [26 (link)] at 10 μM final concentration, for 48 hours.