Figure 5.

Cyclin E2/CDK2 localises to chromatin of endoreduplicating cells, and cyclin E2 depletion abrogates endoreduplication. (A) HeLa cells synchronised with thymidine were treated for 3 h with deoxycytidine (d-CT), subsequently treated with RO3306 and were then monitored by flow cytometry for the commencement of endoreduplication. (B) DNA replication was confirmed by BrdU incorporation, where cells were pulsed with BrdU at 24 h post RO3306 addition for 15 min. N = sets of chromosomes. (C) Cytosolic, nuclear soluble and chromatin lysates were collected at intervals following RO3306 addition, and western blotted for the preRC proteins Cdt1, Cdc6, MCM2, MCM7 and for cyclin E1, cyclin E2, CDK2 and GAPDH. TCL = total cell lysate. The uncropped western blot figure in Figures S5–S7. (D) The relative proportion of protein in each cell fraction was quantitated by ImageJ, and compared with a chi-squared test. (E) Purified chromatin lysates were immunoprecipitated with IgG, cyclin E1 or cyclin E2 and immunoprecipitates were western blotted for preRC proteins and CDK2. The uncropped western blot figure in Figures S8 and S9. (F) HeLa cells were transfected with siRNAs to Ddb1 and a non-targeting control or siRNAs to cyclin E1 or cyclin E2. After 14 h, cells were treated with 10 µM RO3306, and cells were collected 48 h later for analysis for >4N cells by flow cytometry and by western blotting. (G) Western blotting of siRNA-treated cells for Ddb1, Cdt1, cyclin E1, cyclin E2 and GAPDH. (H) Quantitation of >4N cells following siRNA/RO3306 treatment. Treatments were compared by one-way ANOVA, followed by Tukey’s multiple comparisons. The uncropped western blot figure in Figure S10.