Measurement of the labile iron pool (LIP)

The labile iron pool was measured essentially as described [72]. Briefly, cells were transfected with siRNA or treated with doxycycline for 48 hours. Cells were then transferred to 96-well plates and incubated for an additional 24 hours in growth medium with or without 200 μM ferric ammonium citrate (Sigma-Aldrich, F5879) for 4 or 24 hours prior to assay. Cells were washed, incubated with 2 μM calcein acetoxymethyl ester (Life Technologies, Carlsbad, CA, USA, C1430) for 15 to 30 minutes at 37 °C, washed with phenol-free EMEM, and 100 μM starch-conjugated deferoxamine (DFO) was added (a generous gift of Biomedical Frontiers, Inc., Minneapolis, MN, USA). Fluorescence was measured at 485 nm excitation and 535 nm emission (BioTek Synergy 2, BioTek, Winooski, VT, USA). Following stabilization of the fluorescence signal, 10 μM salicylaldehyde isonicotinoyl hydrazone (SIH) was added for several minutes until a stable signal was obtained. The change in fluorescence following the addition of SIH (ΔF) was used as a measure of the labile iron pool.