In situ RT-PCR for localization of p21^CIP1/WAF1 mRNA demonstrates that its nuclear translocation to the cytosol is partially inhibited after Fe chelation with DFO or 311. MCF-7 cells were grown on slides and incubated with control medium (Con), 311 (25 μM), DFO (250 μM), or Act D (5 nM) for 24 hours at 37°C. Cells were then fixed, and in situ RT-PCR was performed. Visual assessment of 300 cells was performed to examine cytosolic or nuclear localization of p21^CIP1/WAF1 mRNA (dark green/brown stain), and the percentages were calculated (***P < .001 relative to the control). (A) Con; (B) Act D–treated cells; (C) 311-treated cells; and (D) DFO-treated cells. (E) Estimation of the nuclear and cytosolic localization of p21^CIP1/WAF1 mRNA by cell counts (n = 300). Results are presented as means ± SD. Images in panels A-D were taken by Olympus microscope BX51 with UPLFLN 40× objective (0.75 numeric aperture) and attached DP30BW digital camera (Olympus, Tokyo, Japan). Images were analyzed using NIH Image J program (Bethesda, MD).