FRDA fibroblasts have impaired iron uptake, accumulate TfR1 at plasma membrane, and exhibit increased transferrin endocytosis. (A) Immunoblots of proteins involved in iron homeostasis, ROS defense, and mitochondrial function in control (C1-C3) and FRDA (P1-P5) cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Immunoblotting quantifications are presented in supplemental Figure 3. (B) Examples of TfR1 labeling in fibroblasts of control (C1-C3) and FRDA (P1-P5) fibroblasts. Cell analysis was based on Hoechst⁺ signals. Scale bar, 10 µm. (C) Quantification of membrane-bound TfR1 signal in control (C*) and FRDA (P1-P5) fibroblasts grown in regular medium, using IDEAS software (Amnis Corporation). (D) Basal fluorescence intensity of Alexa 555–Tf signal in control (C*) and FRDA (P1-P5) fibroblasts after 5 minutes of Alexa 555–Tf incubation. (E) Relative mean fluorescence intensity of Alexa 555–Tf signal for 40 minutes, after 30 minutes of incubation, for control (C*) and FRDA (P1-P5) fibroblasts. Number of cells acquired was >15 cells per experiment. All bar plots show mean ± standard error (n = 3). Analyses of variance and Student t tests were used to compare patients’ values with control mean. *P < .05, **P < .01, ***P < .001.