*4.7. Western Blot Analysis*

Cells were harvested, lysed with cell lysis buffer (50 mM Tris-Cl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and protease inhibitor) for 20 min and centrifuged at 13,000 rpm (4 ◦C) for 20 min. Twenty micrograms of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Protran nitrocellulose membrane, Whatman, UK). The membrane was blocked with 5% nonfat milk, probed with specific primary antibodies, incubated with HRPconjugated secondary IgG antibodies (Calbiochem, San Diego, CA, USA), and visualized using an enhanced chemiluminescence detection system (Amersham ECL kit, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Antibodies against cleaved caspase-8, -3, and -9; GAPDH; p38; phospho-Akt; phospho-mTOR; phospho-PI3K; and total Akt were obtained from Cell Signaling (Danvers, MA, USA). Antibodies against actin, Bax, Bcl-2, Beclin, PARP/p85, phospho-Erk, phospho-p38, total Erk, and total mTOR were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-PI3K antibody was obtained from Merck Millipore (Burlington, MA, USA). The anti-LC3 antibody was obtained from Novus Biologicals (Centennial, CO, USA). The anti-p62 antibody was obtained from Abcam (Cambridge, UK).

#### *4.8. Quantification of Autophagy*

Autophagy was quantified by counting the percentage of cells showing the accumulation of GFP-LC3 in vacuoles. Cells presenting a mostly diffuse distribution of GFP-LC3 in the cytoplasm and nucleus were considered non-autophagic, whereas cells representing several intense punctate GFP-LC3 aggregates with no nuclear GFP-LC3 were classified

as autophagic. Cells were fixed with paraformaldehyde (4% *w*/*v*) for GFP-LC3 and immunofluorescence assays. Images were acquired using confocal microscopy (Carl Zeiss, Oberkochen, Germany).
