Cellular iron mobilization and/or ferritin iron decrements with ELT from HuH7, H9C2, and RINm5F cells. (A) Dose response for iron release from HuH7 cells at 8 hours is shown. (B) Dose response for iron release from H9C2 cells at 1, 2, 4, and 8 hours is shown. Cells were loaded with iron, as described in “Materials and methods.” Adherent cells were rinsed 4 times, including 1 wash containing DFO at 30 µM IBE and 3 PBS washes, and subsequently exposed to ELT and other chelators for the times shown. Chelator-containing supernatants were then removed, and the cells washed 4 times as described before lysing with 200 mM NaOH. Intracellular iron concentration was then determined at each point, using the ferrozine assay described in “Materials and methods” and normalized for total cellular protein in each well. Results shown are expressed as the percentage of T₀ cellular iron released at the times shown and are the mean ± SEM of 6 replicates in 1 representative experiment. (C) Iron release by ELT 10 μΜ and DFO, DFP, and DFX 10 μΜ IBE after 8 hours of treatment in RINm5F cells and (D) iron release by ELT, CP40 and a combination of ELT and CP40 in HuH7 cells. Cells were iron-loaded using two 10-hour changes of 10% FBS-containing RPMI media and rinsed as above. Comparison of the effect of ELT on ferritin and total cellular iron mobilization in (E) HuH7 hepatocyte and (F) H9C2 cardiomyocyte cells is shown. After iron loading, chelator treatment for 8 hours, and rinsing, iron content was ascertained as described earlier. Ferritin was quantified using commercially available enzyme-linked immunosorbent assay kits appropriate for our rat and human cell lines. Results are expressed as the percentage of T₀ cellular iron mobilized or decrement of ferritin expressed as percentage of T₀ values at the times shown and are the mean ± SEM of 3 replicates of 1 representative experiment.