S were subsequently washed and resuspended in phosphate-citrate buffer. After 30 min

S were subsequently washed and resuspended in phosphate-citrate buffer. After 30 min, DNA was stained with 25 mg/ml PI in a reaction solution containing 50 mg/ml RNAse A for 30 min at 37 1C in the dark. Fluorescence emitted from the PIDNA was measured for individual cells using a FACS flow cytometer. Data from three experiments were collected and mean and s.d. of the percentage of cells in the G2/M phase were calculated. Western blot analysis of H3 phosphorylation, Haspin, cyclin B1 and BUB1 expression HCT-116, HeLa and MDA-MB-231 cells were treated with 500 nM of CHR-6494 compound for 48 h, and then harvested and washed twice with PBS. Finally, they were centrifuged at 400 g for 5 min. Cell pellets were resuspended in lysis SB-203580 buffer, sonicated and boiled for 5 min. Immunofluorescence staining HeLa, HCT-116 and MDA-MB-231 cells were grown on coverslips and, after treatment, fixed with freshly prepared 4% paraformaldehyde for 20 min at RT or with 100% cold CF-101 site methanol for 10 min at 20 1C. Cells were mildly permeabilized with PBS containing 0.1% Triton and 2% bovine serum albumin at RT for 1 h. Thereafter, incubation with primary antibodies diluted in PBS with 0.1% bovine serum albumin was performed for 1 h monoclonal antibody, Millipore, 04746; a-tubulin monoclonal antibody, Sigma, T6199; g-tubulin monoclonal antibody, Sigma, T6557; anti-phospho-histone H3, Millipore, 06570), washed intensively and incubated with adequate secondary antibodies labeled with Alexa 488 and Alexa 568 dyes. After staining, coverslips PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19855441 were mounted in Mowiol with DAPI. Images were acquired using a confocal spectral Leica SP5 microscope. Z-projection and image processing were performed using ImageJ software. Chicken embryo aortic arch assay We performed the chicken embryo aortic arch assay as described previously. Aortic rings of B0.8 mm length were prepared from the five aortic arches of 13-day-old chicken embryos. Each aortic ring was placed in the center of a well in a 48-well plate, covered with Matrigel and incubated with bFGF or bFGF and CHR-6494 at either 500 nM or 1.0 mM concentration, or in the presence of medium alone as a negative control. The plates were incubated at 37 1C in 5% CO2 for 24 to 36 h, and microvessels sprouting from each aortic ring were photographed under an inverted microscope. The surface covered by the new vessels was quantified by image analysis. Six independent rings were measured per treatment. The KruskalWallis test was used to identify statistically significant differences among groups. A small molecule inhibitor of Haspin D Huertas et al 1417 Xenografted nude mice Athymic nu/nu male mice, aged 4 5 weeks, were used for tumor xenograft assays. Animals were maintained in a sterile environment; their cages, food and bedding were sterilized by autoclaving. The experimental design was approved by the Bellvitge Biomedical Research Institute animal facility committee. Mice were anesthetized and tumor cells were injected subcutaneously. In all, 3.5 106 exponentially growing HCT-116 cells diluted in 250 ml of sterile PBS were injected subcutaneously in each animal. Body weight was recorded and tumor dimensions were measured twice weekly using digital calipers. Tumor volume was estimated according to the formula V D d2/2, where D is the long axis and d the short axis of tumor. When tumors reached an average volume of 200 mm3, 24 mice harboring homogeneous tumor sizes were randomized into two groups: control group treated with vehicle; treatmen.S were subsequently washed and resuspended in phosphate-citrate buffer. After 30 min, DNA was stained with 25 mg/ml PI in a reaction solution containing 50 mg/ml RNAse A for 30 min at 37 1C in the dark. Fluorescence emitted from the PIDNA was measured for individual cells using a FACS flow cytometer. Data from three experiments were collected and mean and s.d. of the percentage of cells in the G2/M phase were calculated. Western blot analysis of H3 phosphorylation, Haspin, cyclin B1 and BUB1 expression HCT-116, HeLa and MDA-MB-231 cells were treated with 500 nM of CHR-6494 compound for 48 h, and then harvested and washed twice with PBS. Finally, they were centrifuged at 400 g for 5 min. Cell pellets were resuspended in lysis buffer, sonicated and boiled for 5 min. Immunofluorescence staining HeLa, HCT-116 and MDA-MB-231 cells were grown on coverslips and, after treatment, fixed with freshly prepared 4% paraformaldehyde for 20 min at RT or with 100% cold methanol for 10 min at 20 1C. Cells were mildly permeabilized with PBS containing 0.1% Triton and 2% bovine serum albumin at RT for 1 h. Thereafter, incubation with primary antibodies diluted in PBS with 0.1% bovine serum albumin was performed for 1 h monoclonal antibody, Millipore, 04746; a-tubulin monoclonal antibody, Sigma, T6199; g-tubulin monoclonal antibody, Sigma, T6557; anti-phospho-histone H3, Millipore, 06570), washed intensively and incubated with adequate secondary antibodies labeled with Alexa 488 and Alexa 568 dyes. After staining, coverslips PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19855441 were mounted in Mowiol with DAPI. Images were acquired using a confocal spectral Leica SP5 microscope. Z-projection and image processing were performed using ImageJ software. Chicken embryo aortic arch assay We performed the chicken embryo aortic arch assay as described previously. Aortic rings of B0.8 mm length were prepared from the five aortic arches of 13-day-old chicken embryos. Each aortic ring was placed in the center of a well in a 48-well plate, covered with Matrigel and incubated with bFGF or bFGF and CHR-6494 at either 500 nM or 1.0 mM concentration, or in the presence of medium alone as a negative control. The plates were incubated at 37 1C in 5% CO2 for 24 to 36 h, and microvessels sprouting from each aortic ring were photographed under an inverted microscope. The surface covered by the new vessels was quantified by image analysis. Six independent rings were measured per treatment. The KruskalWallis test was used to identify statistically significant differences among groups. A small molecule inhibitor of Haspin D Huertas et al 1417 Xenografted nude mice Athymic nu/nu male mice, aged 4 5 weeks, were used for tumor xenograft assays. Animals were maintained in a sterile environment; their cages, food and bedding were sterilized by autoclaving. The experimental design was approved by the Bellvitge Biomedical Research Institute animal facility committee. Mice were anesthetized and tumor cells were injected subcutaneously. In all, 3.5 106 exponentially growing HCT-116 cells diluted in 250 ml of sterile PBS were injected subcutaneously in each animal. Body weight was recorded and tumor dimensions were measured twice weekly using digital calipers. Tumor volume was estimated according to the formula V D d2/2, where D is the long axis and d the short axis of tumor. When tumors reached an average volume of 200 mm3, 24 mice harboring homogeneous tumor sizes were randomized into two groups: control group treated with vehicle; treatmen.