Protein stability

　　Protein stability assays employing 50 μg/ml cycloheximide (CHX) (Sigma) to inhibit de novo protein synthesis were performed as described.LED Candle Lights 25 Cell lysates were subjected to SDS-PAGE and immunoblotting with an anti-survivin antibody (Abcam). For proteasome inhibition, 25 μM of MG132 (Calbiochem) was added 1 h before CHX. For autophagy-dependent protein degradation assay, 5 mM 3-MA was added 4 h before CHX.

　　Immunoprecipitation assays

　　Cell lysates containing equal amounts of protein (200–400 μg) were immunoprecipitated with anti-survivin antibody (Abcam) overnight at 4 °C. Immune complexes were precipitated with protein A/G-agarose beads (Sigma-Aldrich) for 4 h at 4 °C. Immunoprecipitates were separated by SDS-PAGE and immunoblotted.

　　Immunocytochemistry

　　hASCs grown on coverslips were fixed with 4% (w/v) paraformaldehyde, rehydrated with 2% (v/v) fish skin gelatin and permeabilized with 0.2% Triton X-100 prior to incubation with 5% (v/v) goat serum. Subsequently, cells were incubated overnight at 4 °C with anti-cleaved caspase-3 antibody (Cell Signaling Technology) in PBS containing 1% goat serum. Coverslips were washed with PBS and incubated for 1 h at room temperature with 1:100 Alexa Fluor 488 (Life Technologies, Carlsbad, CA, USA) followed by mounting with Pro Long Gold Antifade Reagent with 40,6-diamidino-2-phenylindole, DAPI (Invitrogen). Images were acquired on a Leica DM 4000B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and captured with a Leica DFC 300 FX camera (Leica Microsystems).

　　Cell apoptosis flow cytometry assay