FIGURE 4.

Abrogation of NF-YA expression using shRNA markedly decreases GADD45α mRNA and protein expression after incubation with chelators relative to the scrambled shRNA control (Scrambled). In addition, the expression of NF-YA is regulated by cellular iron levels. A, in MCF7 cells transfected with the shRNA plasmids (ID#1–2), the levels of NF-YA and GADD45α mRNA is much lower than in cells transfected with the negative control plasmid (i.e. scrambled shRNA). As a result of the reduced expression of NF-YA, the up-regulation of GADD45α mRNA is compromised after incubation with DFO (250 μm) or 311 (25 μm) for 24 h/37 °C. B, Western analysis confirms the results from RT-PCR in A showing that in MCF7 cells transfected with NF-YA shRNA relative to scrambled shRNA, the expression of NF-YA is markedly reduced. Consequently, the up-regulation of GADD45α expression is abrogated after incubation with DFO (250 μm) or 311 (25 μm) for 24 h/37 °C. C, to further examine the regulation of NF-Y expression by iron levels, MCF7 cells were incubated with either control medium, or this medium containing DFO (250 μm) or 311 (25 μm) for 24 h/37 °C. This medium was then removed and the cells reincubated with either control medium, or this medium containing DFO (250 μm), 311 (25 μm) or the iron donor, ferric ammonium citrate (FAC; 100 μg/ml). Results shown are typical gels from three experiments, while the densitometry is mean ± S.D. **, p < 0.01; ***, p < 0.001, relative to the control.