Figure 2. Cellular responses to CCT020312 treatment.

A) Concentration-dependence of P-S608-pRB loss in CCT020312-exposed cells. HT29 cells seeded in 96-well plates were exposed to CCT020312 for 24 hours. Ser608 pRB phosphorylation was quantified using the cell-based immunoassay for the detection of pRB-P-Ser608 as employed for the primary screen (Barrie et al. 2003). Signals normalized to protein content (BCA assay) are shown. Error bars represent the standard error of the mean (n−3). The range for linear response is indicated. B) C) Effects of CCT020312 on cell cycle progression and DNA synthesis. HT29 cells were treated for 16 and 24 hours with 10 µM CCT020312. Cells were stained with propidium iodide and analysed by flow-cytometry (B). Cells were treated with CCT020312 for 16 or 24 hours. BrdU was added to the medium for the final two hours. Cells were stained with anti-BrdU antibody and analysed by flow-cytometry (C). D) Accumulation of Ser608 unphosphorylated pRB in CCT020312 exposed cells. HT29 cells were incubated in the presence of the vehicle (MOCK) or CCT020312 for 24 hours and analysed by immunoblotting. NP-pRB denotes use of the antibody for detection of the non-phosphorylated Ser608 pRB site. E) Marker expression 24 h post CCT020312 exposure. HT29 cells were exposed to 10 µM CCT020312 or vehicle (MOCK) for 24 hours. Lysates were analysed by immunoblot for marker proteins as indicated. Membrane staining with amido black documents loading. F) CCT020312 induces a rapid loss of D cyclin expression. HT29 cells were treated with 10 µM CCT020312 for the times indicated and lysates analyzed as in E. Tubulin probing documents loading.