Iron chelation paradoxically up-regulates p21^CIP1/WAF1 mRNA levels and down-regulates protein expression. MCF-7 cells were incubated with 311 (25 μM), DFO (250 μM), or Act D (5 nM) for 24 hours at 37°C. Total mRNA and protein were then extracted, and RT-PCR and Western blotting were conducted to detect p21^CIP1/WAF1 mRNA and protein levels, respectively. β-actin was used as a loading control. (A) Effect of DFO, 311, and Act D on p21^CIP1/WAF1 mRNA levels (***P < .001 relative to the control; n = 3). (B) Effect of 311 and DFO on p21^CIP1/WAF1 protein levels (***P < .001 relative to the control; n = 3). Error bars are mean ± SD (standard deviation). (C) p21^CIP1/WAF1 mRNA and (D) p21^CIP1/WAF1 protein expression as a function of incubation time with 311 (25 μM) at 37°C. (E) Effect of 311 and DFO concentration on p21^CIP1/WAF1 mRNA and protein expression after an incubation of 24 hours at 37°C. Results are typical of 3 independent experiments.