γ h2ax

After being irradiated, the cells were incubated for 24 h and then lysed with protease inhibitor. Aliquots of cell lysates were loaded at adequate amounts onto gels and then subjected to electrophoresis. Samples was then transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Burlington, MA, USA) and further incubated with primary antibodies to phosphorylated ATM (pATM, GeneTex, Irvine, CA, USA), poly (ADP-ribose) polymerase-1 (PARP1, Cell Signaling Technology, Danvers, MA, USA), γ-H2AX (GeneTex), and β-tubulin (Merck Millipore, Burlington, MA, USA). Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies followed by enhanced chemiluminescence (ECL, Boehringer Mannheim, Mannheim, Germany). Images were obtained after washing out the secondary antibody. The fluorescence images were visually scanned for luminescence and quantitatively analyzed using ImageJ software.

Total cell extract was prepared by boiling the cells in SDS-sample buffer. Aliquots of the extracts were then analyzed by SDS polyacrylamide gel electrophoresis and western blotting with the following antibodies: Myb (5E11, Sleeman, 1993), β-actin (Sigma-Aldrich, AC-15), p53 (Sigma-Aldrich, DO-1), γ-H2AX (Genetex, GTX61796). Preparation of polyadenylated RNA and northern blotting was performed as described^{31 (link)}. Northern blots were hybridized sequentially with radiolabeled probes derived from cDNA clones of the respective genes and the intensities of the resulting bands were quantified with a phosphor imager.

MPT0G211, tubastatin A (TBA), and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan), and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-Tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA). PARP and CDC2 were from Santa Cruz Biotechnology (Dallas, TX, USA).