Figure 4.

Fe²⁺ uptake in mitochondria from rDMT1-overexpressing HEK293 cells is decreased by the DMT1 inhibitor CISMBI. PGSK-loaded mitochondria (pH 7.0_i) from doxycycline-induced rDMT1-HEK293 cells were assayed for quenching induced by 4 µM Fe²⁺ in the absence or presence of 0.1 mM of the competitive DMT1 inhibitor CISMBI that had been pre-incubated for 10 min prior to addition of Fe²⁺. (A) Representative kinetics of fluorescence changes after addition of 4 µM Fe²⁺ are normalized to the initial fluorescence of the second phase (100%). (B) Statistical analysis of second phase PGSK quenching. Fluorescence quenching (−ΔPGSK) over 450 sec (to account for a slow onset of inhibition), as indicated by the scale bar in A, is plotted as means ± SEM of 12 measurements in 4 independent experiments. −, no CISMBI; + , CISMBI. *P < 0.05. RFU, relative fluorescence units.