Figure 5.

(A) Cyclin A is absent in DFO‐treated neuroblastoma cells but occurs in cells arrested with aphidicolin or hydroxyurea. Serum starved neuroblastoma cells for 24 h were subcultured in CM (FCS), or RPMI/10% FCS with hydroxyurea, aphidicolin or DFO. The dishes were incubated for 24 h and the cells were harvested in 0.5 mL of cold PBS, a 50 μL portion of cells was used for FACS and the rest were centrifuged, supernatant removed and added with 200 μL of hot SDS loading buffer. Forty microliter samples of the lysate were separated by 10% SDS‐PAGE and the proteins were transferred to PVDF membranes. This was probed for cyclin A. Then, the membrane was stripped and was probed for β‐actin. All treatments were conducted in duplicate. (B) Cyclin D1 protein levels are not affected by the DFO block. Confluent neuroblastoma cells were serum starved in medium with DFO. After 24 h, the media was replaced with RPMI/10%FCS with or without DFO. The cells were sampled at time of release from DFO, 8 and 12 h later. Gel electrophoresis was performed as detailed in (A).