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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Mol Cancer Ther. 2013 Jul 9;12(9):1860–1873. doi: 10.1158/1535-7163.MCT-13-0157

Figure 5. Cisplatin plus Chk inhibitor induces polyploidy and subsequent mitotic catastrophe in p53 mutant or p53 knockdown HNSCC cells.

Figure 5

A) Inhibition of Chk1 activity was detected in HN31 cells by measuring the levels of Chk1 autophosphorylation on S296. B) Cell lysates from HN31, HN30, HN30-shp53 cells under individual treatments were collected at 24hrs and the levels of DNA damage response markers and also the levels of phospho-H3 (S10), a mitotic marker were examined by western blot. p21 levels were examined from cell lysates collected at 72 hrs. C) HN30, HN31 and HN30-shp53 cells were treated with DMSO (CNT), AZD7762 alone (Chk 100nM), cisplatin (Cisp 1.5μM), or cisplatin plus AZD7762 (Cisp plus Chk). The duration of individual treatments was similar to that in clonogenic assays. Cells were collected at 24, 48 and 72 hrs, fixed, stained with propidium iodide and FACSCAN analysis was performed to determine %8N or polyploidy values. Each cell cycle experiment was performed at least two times. In some cases, error bars may not be visible because they are smaller than marker symbols. †, significantly greater than cisplatin treatment alone in each group, *, significantly greater than the combination treatment in HN30 cells at 72hrs time point, using one way ANOVA and bonferroni's multiple comparison test. D) HN31 and HN30-shp53 cells were subjected to individual treatments as described above. At four days post-treatment, cells were fixed, stained with DAPI and counterstained with FITC-Phalloidin. Light fluorescence microscopy was performed and representative images under each treatment were taken. Red arrow under combination treatment show multi-nucleated cells. Percentage of multinucleated cells under each treatment condition are plotted on graph. *, significantly greater than cisplatin treatment alone using two tailed student's t test. E) HN31 and HN30-shp53 cells were exposed to individual treatments as above and harvested at 24, 48, 72 and 96hrs, and sub G1 values were quantitated using flow cytomtery. ‡, significantly greater than cisplatin treatment alone in each group using two tailed student's t test.