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Immunohistochemistry was performed as previously described on the xenografted tumours with antibodies specific to TfR1 (1/250), ferritin-H (1/200) and FPN (1/200) (Boult et?al., 2008). Slides were scored for intensity of immunoreactivity and the percentage of epithelial cells stained (Di Martino et?al., 2006). All experimental errors are http://www.selleckchem.com/products/pifithrin-alpha.html shown as two standard errors of the mean (representing 95% confidence intervals). To assess the ability of iron chelators to overcome drug resistance, an iron chelator (Dp44mT, deferasirox or DFO) and a chemotherapeutic agent (cisplatin, 5-FU or epirubicin) were examined in combination. Two series of cell counts were performed: one at various concentrations of the iron chelator alone and another in combination with the chemotherapy agent at a fixed concentration. The additional effect of the iron chelator was estimated from the difference between the two series of counts, and significance was assessed using a paired Student's t-test or Wilcoxon signed rank test. Data were considered statistically significant when P http://www.selleckchem.com/screening/pfizer-licensed-library.html cell models was explored using cellular iron uptake and cellular iron mobilization assays (Figure?1). It should be noted that these assays implement highly sensitive estimation of the radioisotope 59Fe using ��-counting. This enables direct measurement of the effect of the chelators on both iron mobilization and inhibition of iron uptake from 59Fe-Tf. Cells were incubated with 59Fe-Tf with increasing concentrations of DFO and deferasirox (1�C20?��M) to assess their ability to prevent cellular iron uptake from the physiological iron donor, transferrin (Le and Richardson, 2002). Both DFO and the experimental chelator, Dp44mT, were used as positive controls, as their activities are well characterized (Richardson et?al., 1995; Yuan et?al., 2004). Of the three chelators used, Dp44mT exhibited the most profound inhibition of cellular iron uptake, limiting uptake to ?10�C30% of the control in the OE33, OE19 and OE21 cell lines (Figure?1A). Deferasirox inhibited 59Fe uptake to ?20�C50% of the control, http://en.wikipedia.org/wiki/Temsirolimus with DFO exhibiting the weakest inhibition (?50�C90%) across all three lines (Figure?1A). To assess the ability of the iron chelators to mobilize cellular iron, cells were pre-loaded with 59Fe then incubated with increasing concentrations of chelator (1�C20?��M). The amount of 59Fe released was measured in the supernatant and expressed as a percentage relative to the total 59Fe (cellular plus released 59Fe). Across all cell lines, the most effective chelator was Dp44mT, mobilizing ?60�C75% of cellular 59Fe. Deferasirox and DFO were less effective and mobilized ?30�C65% of cellular 59Fe (Figure?1B). Cellular iron levels are mainly regulated at the post-transcriptional level by the iron regulatory proteins 1 and 2 (IRP1 and 2) that homeostatically control iron levels (Muckenthaler et?al., 2008).