Cenp a

After treatment, cells grown on sterile poly-L-lysine (Sigma P4832) coated coverslips were fixed in 100% methanol (6 min, -20°C). Primary antibodies were: alpha-tubulin, either unconjugated (B5-1-2) or FITC-conjugated (DM1A), both from Sigma; Bub1 (Millipore, MAB3610); CENP-A (Abcam, mouse monoclonal 13939); gamma-tubulin (Sigma-Aldrich, GTU-88). Secondary antibodies were conjugated to FITC or Cy3 (Jackson Immunoresearch Laboratories), Texas Red (Vector Laboratories), or AMCA (Santa Cruz Biotechnology). DNA was stained with 0.05 μg/ml DAPI (Sigma D9542) and coverslips were mounted in Vectashield (Vector Laboratories).

Chromosome spreads were prepared by the “drying down technique” as previously described [49 ]. Briefly, ovaries were collected from E16.5 mice and incubated in warmed PBS until their use. Ovaries were then incubated in hypotonic extraction buffer [30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF), pH 8.2] for 30 minutes, then teased apart in 100 mM sucrose. The ovarian single cell suspension was then pipetted onto slides wetted in 1% PFA with 0.2% Triton X-100 and allowed to settle overnight in a humid chamber at 37°C. The next day, slides were air-dried, incubated in 0.4% Photo-Flo (Kodak) for 2 minutes, air-dried again and then either stained or stored at -80°C. Spreads were stained by immunofluorescence as described above using primary antibodies against MLH1 (BD Pharmigen), SYCP1 (Abcam), CENP-A (Abcam), γH2AX (Millipore) or SYCP3 (Santa Cruz). To determine prophase I stage, SYCP3 configuration (as shown in S1 Fig) was used. To determine percent oocyte asynapsis, >20 CENP-A centromeric foci was used as a quantitative assessment. For both analyses, four animals yielding approximately 50 oocytes and 10–20 pachytene oocytes each were utilized. To quantify meiotic recombination, 25 or more pachytene oocytes per animal were scored for MLH1 foci on chromosome cores as visualized by SYCP3 staining.

Human prostate adenocarcinoma cell lines, PC-3 and DU-145, were obtained from American Type Culture Collection (Rockville, MD, USA). RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from GIBCO/BRL Life Technologies (Grand Island, NY). Antibodies of PARP-1, Bcl-2, Bcl-xL, Bak, Mcl-1, α-tubulin, cyclin A, cyclin B, cyclin-dependent kinase (Cdk) 1, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies of cleaved caspase-9, caspase-8, β-tubulin (Alexa Fluor 594 Conjugate), p-Cdk1^Thr161, and p-Cdk1^Tyr15 were from Cell Signaling Technologies (Boston, MA). Stathmin-1, BUBR1, and CENP-A were from Abcam (Cambridge, UK). MPM2 was from Millipore (Bedford, MA, USA). Caspase-3 was purchased from Imgenex (San Diego, CA). Antibody of PDE5 was from OriGene Technologies (Rockville, MD, USA). PDE5 small interfering RNA (siRNA) was from GE Healthcare Dharmacon (Chicago, USA). JC-1 and DAPI were from Molecular Probes (Eugene, OR, USA). Anti-mouse and anti-rabbit IgGs were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Leupeptin, phosphatase inhibitors (NaF and Na₃VO₄), dithiothreitol, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), and all other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA).

HCT116-PLK1 cells were treated with TET induction and/or cantharidin and fixed with 100% cold methanol for 10 minutes and permeabilized in 0.25% Triton X-100. Antibody incubation was carried out in 1% BSA in PBS for 1 hour at 37°C with the following antibodies from Abcam: NEDD1 (abcam57336), β-tubulin (abcam6046), CENPA (abcam13939). The γH2AX antibody (2607372) was obtained from EMD Millipore. Primary antibodies were detected with Alexafluor-594 and Alexafluor-488-conjugated secondary antibodies (Life Technologies, A11005, A11034). DNA was stained using DAPI. Three dimensional image stacks of mitotic cells were acquired in 0.2μm steps using a 63X oil-immersion objective on an FV300 confocal laser scanning biological microscope (Olympus). Image stacks were deconvolved using Fiji software. Centromere distance measurements were made manually using FV10-ASW 4.0 Viewer.

Immunohistochemistry was carried out using standard procedures in the eight TMAs. The sections were stained with antibodies against DAXX (clone ab239806, AbCam, Cambridge, UK, at dilution 1:50), HJURP (clone ab100800, AbCam, at dilution 1:5000), CENP-A (clone Ab217622, AbCam, at dilution 1:200) and H3K4me3 (clone C4C8, Cell Signaling, at dilution 1:1500). Antigen retrieval was performed at pH 6. The Envision (Dako, Agilent, Santa Clara, CA, USA) visualization system was used. DAB (3,3-diaminobenzidine) was used as a chromogen, and hematoxylin as a counterstain. Appropriate positive controls according to the manufacturer were used. As a negative control, the omitted primary antibody and substitution with an irrelevant antiserum was used.
For the purposes of the immunohistochemical evaluation, we calculated the H-score, which serves as a semiquantitative measure of the immunohistochemical protein expression levels. To calculate the H-score, the semiquantitative staining intensity score (score 1 to 3) is multiplied by the percentage of positive cells. Therefore, H-score values range between 0 and 300. The epithelial and the lymphocytic components, as well as the nuclear and cytoplasmic positivity, were separately evaluated.