Artesunate attenuates cellular iron dysregulation in FRDA fibroblasts. (A) Biotin immunoblots correlating with extent of TfR1 palmitoylation in control (C1-C3) and FRDA (P1-P5) fibroblasts grown in regular medium (artesunate⁻) or supplemented with 25 µM of artesunate (artesunate⁺) for 48 hours. For each condition, the topmost panel shows the palmitoylated TfR1 level (immunoprecipitated [IP]: biotin) and the panel below shows the IP amount of TfR1 (IP: TfR1). IP TfR1 was used as a loading control for each condition. Immunoblotting quantifications of palmitoylated and steady-state TfR1 levels are given in supplemental Figure 4A-D. (B) Quantification of membrane-bound TfR1 signal for control (C1-C3) and FRDA (P1-P5) fibroblasts, grown for 48 hours with or without 25 µM of artesunate supplementation, using IDEAS software (Amnis Corporation). Examples of TfR1 labeling in control and FRDA fibroblasts are given in supplemental Figure 3E. (C) Iron quantification using ferrozine-based colorimetric assay in control (C1-C3) and FRDA (P1-P5) fibroblasts grown in regular medium, with or without 25 µM of artesunate for 48 hours. (D) Immunoblot analysis of proteins involved in iron homeostasis and ROS defense in control (C1-C3) and FRDA (P1-P5) fibroblasts grown in regular DMEM medium, with or without 25 µM of artesunate, for 48 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Immunoblotting quantifications are given in supplemental Figure 4F. All bar plots show mean ± standard error (n = 3). Two-way analyses of variance were used to compare untreated with treated values. *P < .05, **P < .01, ***P < .001. ns, nonsignificant.