Figure 2.
Iron accumulation and posttranscriptional regulation. (A) Iron quantification using the ferrozine-based colorimetric assay in control (C1-C3) and FRDA (P1-P5) fibroblasts grown with increasing concentrations of holo-Tf (1.3-13 mM) for 72 hours. (B) Iron quantification for whole cellular extracts of control (C1-C3) and FRDA (P1-P5) fibroblasts grown in FBS-free DMEM medium under low- (FAC−) or high-iron (100 µM of FAC; FAC+) conditions for 72 hours. (C) Relative cytosolic and mitochondrial iron content in control (C*) and FRDA (P1-P5) fibroblasts grown in FBS-free DMEM with 100 µM of FAC for 72 hours. (D) Quantification of mitochondrial ROS by MitoSOX Red staining in control (C*) and FRDA (P1-P5) fibroblasts grown for 72 hours in high-iron medium (100 µM of FAC). (E-F) Posttranscriptional regulation of iron homeostasis in control (C1-C3) and FRDA (P1-P5) fibroblasts grown in FBS-free DMEM under low- (FAC−) or high-iron (FAC+) conditions. TfR1 (TFRC) (E) and ferritin (FTH) (F) messenger RNAs (mRNAs) were quantified by droplet digital polymerase chain reaction and expressed as ratios to GUSB mRNA. (G) IRP1- and IRP2-IRE binding activity determined by EMSA in low- (FAC−) or high-iron (FAC+) condition with or without deferiprone (DFP; 100 µM) using cytosolic extracts from control (C1-C3) and FRDA (P1-P5) cells. Exposure was 300 seconds for the 3 conditions. Equal amounts of fibroblast protein extracts (20 µg) were assayed. Supplemental Figure 2B-C shows evidence for identification of the 2 bands and immunoblotting quantifications. All bar plots show mean ± standard error (n = 3). Multiple Student t tests (A-D) and 2-way analyses of variance (E-F) were used to compare untreated with treated values. *P < .05, **P < .01, ***P < .001. ns, nonsignificant.

Iron accumulation and posttranscriptional regulation. (A) Iron quantification using the ferrozine-based colorimetric assay in control (C1-C3) and FRDA (P1-P5) fibroblasts grown with increasing concentrations of holo-Tf (1.3-13 mM) for 72 hours. (B) Iron quantification for whole cellular extracts of control (C1-C3) and FRDA (P1-P5) fibroblasts grown in FBS-free DMEM medium under low- (FAC−) or high-iron (100 µM of FAC; FAC+) conditions for 72 hours. (C) Relative cytosolic and mitochondrial iron content in control (C*) and FRDA (P1-P5) fibroblasts grown in FBS-free DMEM with 100 µM of FAC for 72 hours. (D) Quantification of mitochondrial ROS by MitoSOX Red staining in control (C*) and FRDA (P1-P5) fibroblasts grown for 72 hours in high-iron medium (100 µM of FAC). (E-F) Posttranscriptional regulation of iron homeostasis in control (C1-C3) and FRDA (P1-P5) fibroblasts grown in FBS-free DMEM under low- (FAC−) or high-iron (FAC+) conditions. TfR1 (TFRC) (E) and ferritin (FTH) (F) messenger RNAs (mRNAs) were quantified by droplet digital polymerase chain reaction and expressed as ratios to GUSB mRNA. (G) IRP1- and IRP2-IRE binding activity determined by EMSA in low- (FAC−) or high-iron (FAC+) condition with or without deferiprone (DFP; 100 µM) using cytosolic extracts from control (C1-C3) and FRDA (P1-P5) cells. Exposure was 300 seconds for the 3 conditions. Equal amounts of fibroblast protein extracts (20 µg) were assayed. Supplemental Figure 2B-C shows evidence for identification of the 2 bands and immunoblotting quantifications. All bar plots show mean ± standard error (n = 3). Multiple Student t tests (A-D) and 2-way analyses of variance (E-F) were used to compare untreated with treated values. *P < .05, **P < .01, ***P < .001. ns, nonsignificant.

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