Cyclin e

Whole cell extracts were lysed in cell lysis buffer (Biosesang, Inc., Seongnam, Korea). Equal quantities of protein (30 µg) were separated on 8–12% SDS-PAGE gels and were subsequently transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Freiburg im Breisgau, Germany). After blocking the membranes with 1% bovine serum albumin and 2% skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the appropriate primary antibody, and were washed three times in phosphate buffered saline with 0.01% Tween-20. The membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. In order to visualize the protein bands, the membranes were treated with enhanced chemiluminescence kit solution (DoGen) and exposed to X-ray film (AGFA Healthcare, Mortsel, Belgium). Anti-PARP, caspase-3, caspase-9, cyclin-dependent kinase (CDK)4, phosphorylated (p-)p53 and p-murine double minute 2 (MDM2) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-CDK1, CDK2, cyclin E, cyclin A, cyclin B, p21, p53 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-cyclin D and Bcl-xL antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

The HeLa cell line (BCRC-60005), C33A cell line (BCRC-60554), DLD-1 cell line (BCRC-60132), LS174T (BCRC-60053), HA22T (BCRC-60168), and HA59T (BCRC-60169) were purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. HeLa, C33A, and LS174T Cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100-U/mL streptomycin, and 100-mg/ml penicillin. The DLD-1 cells were maintained in Roswell Park Memorial Institute medium supplemented with 10% FBS, 100-U/mL streptomycin, and 100-mg/ml penicillin. HA22T and HA59T were maintained in MEM supplemented with 10% FBS, 100-U/mL streptomycin, and 100-mg/ml penicillin (all from Gibco). Primary antibodies targeting the following proteins were used: CDK1, CDK2, cyclin A, cyclin B, cyclin E, p53, and corresponding secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) (all from Santa Cruz); β-actin, ROGDI, p21, γ-H2A.X (phospho S140) and γ-H2A.X (phospho S139) (all from Abcam); and ROGDI (Proteintech, 17047-1-AP).

The monoclonal or polyclonal antibodies against cyclin A, cyclin B, cyclin C, cyclin D, cyclin E, CDK2, CDK4, CDK6, p16, p18 and p27 were purchased from Santa Cruz Biotechnology. The monoclonal antibodies against p21 and p57 were obtained from BD Biosciences. Horseradish peroxidase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. RNA and DNA extract kits, RNA RT and polymerase chain reaction (PCR) kits were obtained from Qiagen. NorthernMAX kit was from Ambion. Immunoblotting was performed using the Enhanced chemiluminescence (ECL) western blot detection kit (Amersham Biosciences). Biotin Chromogenic Detection kit was from Thermo Scientific. Protein A-Sepharose 4B Conjugate and Dynabeads MyOne Streptavidin C1 magnetic beads were obtained from Invitrogen. The cell lines used in this study were from American Type Culture Collection (ATCC).