Hydroxyurea increases Chk1 activation but not thermostabilization by V158411.

<p>(A) A sealed plate containing HT29 cells was submerged and heated in a water bath pre-heated to 37–55°C. Nuclear Chk1 intensity was determined by immunofluorescent staining using the EP691Y antibody and high content imaging. Values are the mean of 3 wells ± SD. (B) HT29 cells were treated with 0 or 2.5 mM HU for 18 hours followed by 0–10 μM V158411 for 30 minutes. Protein expression levels were determined by immunoblotting. (C) HT29 cells were treated with 0 or 2.5 mM HU for 18 hours and then stained with 1 μg/ml Hoechst 33342 for 30 minutes. Cells were imaged on the Operetta and the total nuclear DNA fluorescent intensity plotted against cellular area. Red box, arrested cells. (D) HT29 cells were treated as above. Cells were heated as indicated and nuclear Chk1, pChk1 (S317) or pChk1 (S345) intensity was determined by single cell immunofluorescent imaging. Values are the mean of 3 wells ± SD.