Polyacrylamide gels

Leukemia cells (HL-60, NB4, THP-1 and U937) were seeded in 24-well plates at 5 × 10⁵ cells/well in which E5 was added at different concentrations for incubation of 24 h. Collected and washed with PBS, cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) supplemented with protease inhibitors (Sigma-Aldrich, USA) on the ice for 30 min. The lysates were clarified by centrifugation at 15000 rpm for 10 min. Equal protein (30 µg) was loaded and separated on 12% polyacrylamide gels (Applygen, Beijing, China), and transferred to PVDF membranes (0.2 µm; Millipore, Bedford, MA). Cleaved caspase-3 and β-Actin were probed with specific primary antibodies (Cell Signaling Technology, Beverly, MA) and secondary antibodies conjugated to HRP (Zhongshan Goldenbridge biotechnology Co, Beijing, China). The immunocomplex on the membrane was visualized using Image Quant LAS 4000 (GE Healthcare) with Immobilon ® Western HRP Substrate luminol reagent and peroxide solution (Millipore, Billerica, MA).

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).