Cyclin D1 is down-regulated in confluent RCCs expressing pVHL, and both cyclin D1 and pVHL are down-regulated after Fe chelation. (A) Cyclin D1 is down-regulated in confluent cultures of human RCCs expressing pVHL. Western blot analysis of cyclin D1 and pVHL expression using confluent cultures of human parental RCCs (786-O); these cells either transfected with the VHL hyperexpression vector (WT-8) or this vector without the VHL insert (PRC3). (B) DFO and 311 down-regulate cyclin D1 protein regardless of pVHL status. The 786-O, WT-8, and PRC3 cells under partially confluent culture conditions were incubated with media alone (control) or media containing DFO (250 μM) or 311 (25 μM) for 24 hours. Western blot analysis was performed to detect cyclin D1 and pVHL expression. (C) DFO and 311 down-regulate pVHL and cyclin D1 but up-regulate TfR1 in a variety of cell types. MCF-7 breast cancer cells, SK-N-MC neuroepithelioma cells, and SK-Mel-28 melanoma cells were incubated with either media alone or media containing 311 (25 μM) or DFO (250 μM) over 24 hours, and Western analysis was performed. Results shown are a typical experiment from 3 performed.