Survivin

U87 cells were washed with ice-cold PBS before lysis in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Cells were scraped off from six-well plates and agitated at 4°C for 10 min. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were mixed at 1∶1 ratio with 2× Laemmli buffer. Protein lysates were heated at 95°C for 5 min, separated by 10% Mini-PROTEAN gels (Bio-Rad), transferred onto 0.45 µm nitrocellulose membranes, and probed with antibodies against p-STAT1 (Y701), total STAT1, NF-κB p65, p-Akt, total Akt, p-mTOR, total mTOR, PKR (Cell Signaling), VSV, GFP, NF-YA, NF-YB, NF-YC, p-p70 S6 kinase, total p70 S6 kinase, PI3K, p21, Rb, caspase-3, caspase-9, XIAP, survivin, β-tubulin (Santa Cruz), acetylated H3 (Millipore), and total histone H3 (Abcam). Secondary incubation was performed with anti-goat, anti-rabbit, or anti-mouse IgG HRP-linked antibodies (Cell Signaling). Proteins were visualized using HyGlo chemiluminescent HRP antibody detection reagent (Denville Scientific Inc.) and a Fuji LAS-3000 cooled CCD camera (FUJIFILM).

Human colon cancer cells HT-29 (Cat# 300215) and CaCo-2 (Cat # 300137) were purchased from CLS (cell lines service, Germany). Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 5% CO₂, 37°C and 95% humidity. 3-methyladenine (3-MA) and Z-VAD-FMK were obtained from sigma-Aldrich. Antibodies against target proteins used in this study are: caspase 8, caspase 7, LC3 and Beclin-1 (Cell Signaling, USA); cleaved caspase 3, Cyclin B1, H3 phospho-Ser10, γH2AX (Millipore), TNFα, p62/SQSTMI and cleaved PARP (Abcam), survivin and β-actin (Santa Cruz Biotechnology).

1-(2-Hydroxy-5-methylphenyl)-3-pheyl-1,3-propanedione (HMDB) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). The purity of the compound is >97% by high performance liquid chromatography (HPLC), and dissolved in dimethyl sulfoxide (DMSO). Propidium iodide, RNaseA, and 3-methyladenine (3MA) were available from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against β-actin, Bcl-2, PCNA, and survivin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against LC3A/B, phospho-AMPK (Thr172), AMPK, phospho-Akt (Ser473), Akt, phospho-mTOR (Ser2448), mTOR, phospho-Raptor (Ser792), Raptor, phospho-p70 S6K (Thr389), p70 S6K, and all G1 cell cycle regulators were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Beclin-1 and SQSTM1/p62 were purchased from GeneTex, Inc. (Irvine, CA, USA).