FIG. 7.

Partial depletion of Nfs1 activates endogenous IRP1 for IRE binding. HeLa cells were infected with VSVG-pseudotyped retroviral particles containing pSUPERretro-Nfs1 or empty pSUPERretro vector. (A) Two weeks postinfection, the expression of mitochondrial Nfs1 (top) and β-actin (bottom) was examined by immunoblotting. (B) Transduced cells were left untreated or exposed overnight to 100 μM DFO, and IRE-binding activity was analyzed by EMSA with a ³²P-labeled IRE probe in the absence (top) or presence (bottom) of 2% 2-mercaptoethanol (2-ME). IRE/IRP complexes are indicated by arrows. (C) IRE/IRP band intensities from three independent experiments (means ± SD) were quantified by phosphorimaging and plotted on the right, following normalization with the respective values obtained with 2-mercaptoethanol. (D) Crude cytoplasmic lysates were analyzed for aconitase activity; values correspond to means ± SD from triplicate samples. (E) The growth rate of transduced cells was monitored by direct cell counting. Similar results were obtained with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide proliferation assay (data not shown). *, P < 0.05; **, P < 0.01 (Student's t test).