Caspase-3 Signaling Analysis of E5 Treatment

After being treated with different concentrations of E5 (1–100 μM) for 24 h, cells were collected and subjected to caspase-3 signaling analysis. The western blot was performed as described previously.^{21 (link)} Briefly, cells were lysed in RIPA lysis buffer with protease and phosphatase inhibitors at 4 °C for 30 min. The lysates were centrifuged for 15 min at 12 000 r.p.m. The proteins were loaded and resolved on 12% polyacrylamide gels (Applygen, Beijing, China) and electroblotted to PVDF membranes (0.45 μm, Millipore, Bedford, MA, USA). The blots were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h and probed with anti-cleaved caspase-3 antibody in 5% non-fat milk in TBST overnight at 4 °C. After being washed with TBST, the blots were incubated with the corresponding secondary antibodies. After a final wash, the antibody-antigen complexes on the blots were detected using Image Quant LAS 4000 (GE Healthcare Life Science, Pittsburgh, PA, USA).