Fig. 5

Fig. 5 Targeting mitochondrial ROS improves cell adhesion and reduces podocyte permeability. (a) Adherence of CTNS?/?#1 and CTNS?/?#2, normalized to WT podocytes. Each dot represents the relative number of podocytes attached in one well. (n???3 biological experiments, n???3 technical replicates). Statistical analysis: linear mixed model. (b) Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) fluorescence quantification in CTNS?/?#1 and CTNS?/?#2, normalized to WT. (n???6 biological experiments, n?=?4 technical replicates). Statistical analysis: Dunnett?s One-Way ANOVA. (c) Adherence of shCTNS#1 and shCTNS#2, normalized to shCTR. Each dot represents the relative number of podocytes attached in one well. (n???3 biological experiments, n???3 technical replicates). Statistical analysis: linear mixed model. (d) FITC-BSA fluorescence quantification in shCTNS#1 and shCTNS#2, normalized to shCTR. (n???7 biological experiments, n?=?4 technical replicates. Statistical analysis: Dunn?s Kruskal?Wallis. (e, f, g, h) Adherence of cells incubated with 100 µM cysteamine, 10 µM MitoTEMPO, or 100 µM cysteamine?+?10 µM MitoTEMPO for 48 h, normalized to e. CTNS?/?#1, f. CTNS?/?#2, g. shCTNS#1, and h. shCTNS#2. (n???3 biological experiments, n???3 technical replicates). Statistical analysis: linear mixed model. (I, j, k, l) FITC-BSA fluorescence quantification of cells incubated with 100 µM cysteamine, 10 µM MitoTEMPO, or 100 µM cysteamine?+?10 µM MitoTEMPO for 48 h, normalized to i. CTNS?/?#1, j. CTNS?/?#2, k. shCTNS#1, and l. shCTNS#2. (n???4 biological experiments, n???3 technical replicates). Statistical analysis: Dunnett?s One-Way ANOVA. (m) Millifluidic system to assess podocyte dynamic permeability. Podocytes were seeded in a bioreactor and perfused with media. The figure was created using biorender.com. (n, o) Quantification of FITC-BSA fluorescence intensity in the upper chamber medium after perfusion in n. WT, CTNS?/?#1?+?100 µM cysteamine?+?10 µM MitoTEMPO, and o. shCTR, shCTNS#2?+?100 µM cysteamine?+?10 µM MitoTEMPO for 48 h. (n???3 biological experiments, n?=?4 technical replicates). Statistical analysis: Dunnett?s One-Way ANOVA. Unless noted, each dot represents a biological experiment