Fig. 3

Cystinosis podocytes show an increased mitochondrial glutathione redox state and elevated lipid peroxidation. (a, b) In situ quantification of the mitochondrial glutathione redox potential in a. CTNS−/−#1 and CTNS−/−#2 normalized to WT podocytes, b. shCTNS#1 and shCTNS#2 normalized to shCTR. (n ≥ 4 biological experiments, where each dot represents the average ratio calculated from 10 measurements within a single cell). Statistical analysis: Dunnett’s One-Way ANOVA. AU = arbitrary unit. (c, d, e, f) Superoxide anion levels measured using the mitochondrial superoxide indicator MitoSOX in c. CTNS−/−#1 normalized to WT podocytes and protein content, d. CTNS−/−#2 normalized to WT podocytes and protein content, e. shCTNS#1 normalized to shCTR and protein content, f. shCTNS#2 normalized to shCTR and protein content. (n = 3 biological experiments, n ≥ 5 technical replicates) Statistical analysis: linear mixed model. Each dot represents a technical replicate. (g, h) Left panel: Representative western blot image of Superoxide Dismutase 2 (SOD2) in g. WT, CTNS−/−#1, and CTNS−/−#2, and h. shCTR, shCTNS#1, and shCTNS#2. β-Actin was used as a loading control. Right panel: Quantification of SOD2 protein expression relative to β-Actin. (n = 4 biological experiments, n = 1 technical replicate). Statistical analysis: one-sample t-test, with WT set as the reference. AU = arbitrary units. (i, j, k, l) Ratio of the percentages of BODIPY-C11 + cells measured by flow cytometry in i. CTNS−/−#1 normalized to WT, j. CTNS−/−#2 normalized to WT, k. shCTNS#1 normalized to shCTR, and l. shCTNS#2 normalized to shCTR. (n ≥ 4 biological experiments, n = 1 technical replicate). Statistical analysis: Welch’s unpaired t-test. Unless noted, each dot represents a biological experiment