RT-PCR and real-time RT-PCR RT-PCR and real-time RT-PCR analysis were performed as described previously [24]. The primers and

probes for RT-PCR and the real-time RT-PCR were designed with Primer Express v 2.0 (Applied Biosystems, Inc.) and provided in Table 1. Table 1 Primer Sequences Used for Reverse Transcription-PCR and Real-time Quantitative RT-PCR (5′ to 3′)   Gene Forward primer Reverse primer Probe RT-PCR CENP-H TGCAAGAAAAGCAAATCGAA ATCCCAAGATTCCTGCTGTG     GAPDH CCACCCATGGCAAATTCCATGGCA TCTAGACGGCAGGTCAGGTCCAC   Real-time PCR CENP-H CCTTATTTTGGGGAGTAAAGTCAAT ACAAATGCACAGAAGTATTCCAAAT FAM-TTCCTTAAGGGCAGGATCCT-TAMRA   GAPDH GACTCATGACCACAGTCCATGC AGAGGCAGGGATGATGTTCTG Selleckchem LY294002 FAM-CATCACTGCCACCCAGAAGACTGTG-TAMRA Full gene names: CENP-H, centromere protein H;GAPDH, glyceraldehyde-3-phosphate dehydrogenase Western blot Western blot analysis was performed as described previously[15, 24] using anti-CENP-H (Bethyl Laboratories, Montgomery, Texas, USA), anti-α-Tubulin (Sigma, Saint Louis, Michigan, USA), anti-p21, anti-p27 and anti-Rb antibodies (Cell Signaling, Danvers, Massachusetts, USA). Immunohistochemical analysis The staining procedures and result measure of CENP-H were done as described previously[15, 24]. The cells at each intensity of staining

AZD4547 chemical structure were recorded on a scale of 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellowish brown), and 3 (strong staining = brown). An intensity score of ≥ 2 with at least 50% of malignant cells with positive CENP-H staining was used to classify tumors with high expression, and < 50% of malignant cells with nuclear staining TCL or < 2 intensity score classified tumors with low expression of CENP-H. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay Growing cells (5 × 103 per well) were seeded into 96-well plates. Cells were stained with 100 μl sterile MTT dye (0.5 mg/ml, Sigma, St. Louis, Missouri, USA) at each time point, followed by additional incubation for 4 h at 37°C. After removal of the culture medium from each well, 150 μl of dimethyl sulphoxide

(Sigma, St. Louis, MO, USA) was added and thoroughly mixed for 15 min. The optical density was read at 570 nm using a microplate reader (Bio-Rad 3500, Hercules, California, USA), with 655 nm as the reference wavelength. All experiments were performed in triplicate. Colony formation assays Cells were seeded in 6-well plates (1×103 cells per well) and cultured for two weeks. The colonies were fixed with methanol for 10 min and stained with 1% crystal violet for 1 min. Each group of cells was performed in triplicate. Bromodeoxyuridine (BrdU) incorporation and immunofluorescence Cells grown on cover slips (Fisher, Pittsburgh, Pennsylvania, USA) were synchronized by serum starvation (0.5%FBS) for 48 h and then released into serum-containing medium for 4 h.