Anti survivin

Western blot was done as previously described21 (link). Antibodies specific for the following targets were from Santa Cruz (Heidelberg, Germany): BAX, β-catenin, HSP90, histone deacetylase (HDAC)2, PIG3, p53 and p21. Anti-survivin was obtained from Novus Biologicals (Cambridge, UK).

S116836 (chemical structure, Fig. 1A) was rationale designed and synthesized in our lab [26 ]. S116836 was dissolved in DMSO at a stock concentration of 20 mM and stored in aliquots at −20°C. U0126 and LY294002 were purchased from Calbiochem (San Diego, CA). Cycloheximide (CHX) was bought from Sigma-Aldrich (St. Louis, MO). MG132 was obtained from EMD Bioscience (Billerica, MA). Antibodies against poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), pro-caspase3, X-linked inhibitor of apoptosis protein (XIAP) and cytochrome c were from BD Biosciences (San Jose, CA). Antibodies against phospho-PDGFRα (Y1018), phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-AKT (S473), AKT, caspase-8, caspase-9, Bax and phospho-Bim (S69) were from Cell Signaling Technology (Beverly, MA). Antibodies against phospho-STAT3 (Y705), STAT3 and PDGFRα were from EMD Millipore Upstate (Billerica, MA). Anti-Mcl-1 (S-19) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bim was from Stressgen Enzo Life Sciences (Plymouth Meeting, Pennsylvania). Anti-Survivin was from Novus Biologicals (Littleton, CO, USA). Anti-active-caspase-3 and Anti-β-actin were from Sigma-Aldrich (St. Louis, MO). Anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated antibodies were purchased from Pierce Biotechnology (Los Angeles, CA, USA).

For Western blot analysis, cells or exosomal preparations were lysed using lysis buffer (50 mM Tris (pH 7.5), 1% NP40, 0.25% DOC, 150 mM NaCl₂, 1 mM PMSF, 10 μg/ml aprotinin/leupeptin/pepstatin, 20 mM NaF, 0.2 mM EGTA, 1 mM EDTA (pH 8.0), H₂O). For protein concentrations, the BCA assay (Pierce, Rockford, IL) was used. Proteins from exosomes (20-40 μg) were separated using 12% Bis-Tris polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA) and probed using the following antibodies: mouse monoclonal anti-LAMP1 (Abcam, Cambridge, MA), anti-Survivin-2B, anti-Survivin-∆Ex3 (Cell Signaling, Danvers, MA), and rabbit polyclonal anti-Survivin (Novus, Littleton, CO). Secondary antibodies (IR-Dye conjugated) were goat anti-rabbit and goat anti-mouse immunoglobulin (LICOR, Lincoln, NE). Immunoreactive bands were detected using the Odyssey Imaging System (LICOR, Lincoln, NE) and quantified using ImageQuant software.

Total proteins were extracted using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 5 mM EDTA, pH 8.0) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Protein concentration was determined using Bradford Reagent (Sigma-Aldrich, Milano, Italy). Total protein extracts (40 μg) were separated by SDS–PAGE and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). Membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) containing 5% fat-free dry milk and then incubated with anti-Bcl-2 (#2870, Cell Signaling Technologies, Danvers, MA, USA), anti-Survivin (NB500-201; Novus Biologicals, Milano, Italy), anti-p16(Ink4a) (sc-377412, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-LC3 isoform B (NB600-1384, Novus Biologicals, LLC, Littleton, CO, USA), anti-VDAC (#4866, Cell Signaling Technologies, Danvers, MA, USA), anti-Lamin A/C (#2032, Cell Signaling Technologies, Danvers, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies. Proteins were visualized by ECL (Amersham, Piscataway, NJ, USA) according to the manufacturer’s instructions and quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).

Doxorubicin (Dox), 2-propylpentanoic acid (VPA) and sodium butyrate, oxaliplatin (Oxa), 5-fluorouracil (5-FU) were purchased from Sigma-Aldrich (United Kingdom). SAHA (vorinostat) was obtained from Zolinza (Merck, Whitehouse Station, NJ, USA). anti-PUMA was purchased from eBioscience (UK), anti-MDM2 was from Calbiochem, UK, anti-HDAC1, 2, anti-phosphorylated or acetylated P53 and anti-PARP were obtained from Cell Signaling Technology, UK). anti-P53 (Do1) was from GeneTex Inc., USA and anti-Survivin was from Novus Biologicals, USA, and anti-β-actin (Sigma–Aldrich). Anti-histone H3 and anti-H4 (Abcam, Cambridge, UK) or acetylated histone lysine H3K9, H4K12 and H4K16 (Upstate Biotechnology, Billerica, MA, USA). Binding of primary antibodies was detected with a horseradish-peroxidase-conjugated goat anti-mouse (Pierce, Rockford, IL, USA). After repeated washing in phosphate-buffered saline, bands were visualized by enhanced chemiluminescence (ECL Plus) following the manufacturer's instructions (Amersham).