in MP-470 c-kit inhibitor vitro in the same concentration of ZM447439 for 8 hr. ZM447439 was washed out of the media in some groups of oocytes and all groups were allowed to continue maturation in vitro for 10 hr prior to fixation in cold methanol. The spindle was detected with an anti β tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine chromosome misalignment. Data are shown as mean ± SEM from three independent experiments and were analyzed using two way ANOVA. **P < 0.01, ***P < 0.001. E: As in but confocal microscopy was used to determine the stage of meiosis. Data are shown as mean ± SEM from three independent experiments and were analyzed using two way ANOVA. *P < 0.05. SHUDA et al. Page 18 Mol Reprod Dev. Author manuscript, available in PMC 2011 October 5.
NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Figure SU11274 658084-23-2 6. Effect of ZM447439 on chromosome alignment at Met I and Met II. A: GV intact oocytes were treated with concentrations of ZM447439 ranging from 0 to 10 μM for 1 hr in maturation medium containing milrinone, and matured in vitro in the same concentration of ZM447439 for 8 hr prior to fixation in cold methanol. The spindle was detected with an anti β tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine chromosome alignment at Met I. Data are shown as mean ± SEM from three independent experiments and were analyzed using two way ANOVA. *P < 0.05. B: GVintact oocytes were held for 1 hr in maturation medium containing milrinone and matured in vitro for 10 hr, a time at which most oocytes have passed Met I.
Concentrations of ZM447439 ranging from 0 to 10 μM were added to the media and oocytes were allowed to continue maturation in vitro for 8 hr prior to fixation in cold methanol. The spindle was detected with an anti β tubulin antibody and DNA was visualized with DAPI. Confocal microscopy was used to determine chromosome alignment at Met II. Data are shown as mean ± SEM from three independent experiments and were analyzed using two way ANOVA. ***P < 0.001. SHUDA et al. Page 19 Mol Reprod Dev. Author manuscript, available in PMC 2011 October 5. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Figure 7. AURKB rescue of chromosome alignment defect caused by ZM447439.
GV intact oocytes were microinjected with Gfp, Aurka eGfp, Aurkb eGfp, or Aurkc eGfp mRNA and held for 14 hr in medium containing milrinone. These oocytes were treated with 1.5 μM ZM447439 for 1 hr in maturation medium containing milrinone and matured in vitro in the same concentration of ZM447439 for 8 hr prior to fixation in 3.7% paraformaldehyde. DNA was visualized with propidium iodide. Confocal microscopy was used to determine chromosome alignment. Data are shown as mean ± SEM from three independent experiments and were analyzed using Student,s t test. *P < 0.05. SHUDA et al. Page 20 Mol Reprod Dev. Author manuscript, available in PMC 2011 October 5. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript impactjournals/oncotarget/ Oncotarget, August, Vol.
2, No 8 impactjournals/oncotarget 599 Oncotarget 2011, 2: 599 609 Aurora Kinase Inhibition Overcomes Cetuximab Resistance in Squamous Cell Cancer of the Head and Neck Alexander Hoellein1, Anja Pickhard2, Fabienne von Keitz1, Stephanie Schoeffmann1, Guido Piontek2, Martina Rudelius3, Anja Baumgart1, Stefan Wagenpfeil4, Christian Peschel1, Tobias Dechow1, Henning Bier2 and Ulrich Keller1 1 III. Medical Department, Technische Universität München, Munich, Germany 2 Department of Head and Neck Surgery, Technische Universität München, Munich, Germany 3 Institute of Pathology, Technische Universität München, Munich, Germany 4 Institute for Medical Statistics and Epidemiology, Technische Universität München, Munich, Germany Correspondence to: Ulrich Keller, email: ulrich.kellerlrz.tum.de Keywords: Squamous cell cancer of the head and neck, Aurora kinase, EGFR Received: July 18