Cellular Fe depletion decreases cyclin D1 expression and induces a G₁/S arrest. (A) Fe chelators induce a G₁/S arrest and ablate cyclin D1 protein expression. MCF-7 cells were treated with 10% FCS medium alone or medium containing 10% FCS and the Fe chelators (DFO [250 μM] or 311 [25 μM]) for 24 hours at 37°C. (B) Serum starvation induces a G₁/S arrest but does not affect cyclin D1 expression. In addition, G₁/S arrest induced by serum starvation is reversible upon addition of serum but does not markedly affect cyclin D1 expression. Cells were washed twice with PBS and then incubated with either serum-free media or media containing 10% FCS for 24 hours at 37°C. The serum-starved cells were then released into media containing 10% FCS for 16 hours at 37°C. (C) G₁/S arrest induced by serum starvation is not reversible upon addition of 10% FCS and 250 μM DFO for 16 hours. Cells were washed with PBS and then incubated with either serum-free media or media containing 10% FCS for 24 hours at 37°C. The serum-starved cells were then released into media containing 10% FCS and 250 μM DFO for 16 hours at 37°C. Cells were harvested after each step of the synchronization process for flow cytometric and Western blot analysis. The same results were found for 311. Results shown are from a representative experiment from 3 performed.