Abstract 2379 An optimized protocol for silencing the iron transporter DMT1 in human intestinal cells using siRNA for studies of iron uptake routes

The objective of the present study was to develop and optimize a protocol for silencing the translation of the iron transporter DMT1 in human intestinal cells, using small interfering RNA (siRNA) to be able to efficiently investigate iron uptake routes. Together with pharmacological inhibition of endocytosis and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), we showed that iron uptake from ferrous fumarate is mediated by both specific transport by DMT1 and by clathrin-dependent endocytosis. In addition, we observed that the protein expression of GAPDH, often used as loading control in e.g. Western blot analysis, was affected by iron load and thus may not be a good choice in iron carrier studies. To conclude, the optimized protocol yielded a DMT1 protein silencing efficiency of 95% ± 4.4% in non-confluent Hutu-80 cells and 84%± 10.5% in non-confluent Caco-2 cells, whereas it was only 50.4%, ±0.5 % in confluent Hutu-80 cells and 53.6%, ±16.1% in confluent Caco-2 cells. This work was funded by internal resources