Fig. 4.

Podocytes sense lipid peroxyl radicals through the redox sensitive cysteine residues of RhoA. (A-B) Comparison of podocyte migration using the “wound” assay in normal cells, wild type RhoA transduced podocytes and in cells transduced with the mutant C16/20 A RhoA, after lipid peroxide radical exposure (0.8 − 2 µmol/min, 4 h). Number of cells migrating into the wound was counted in each group in duplicate experiments after 72 h. While normal or Wt podocytes have significantly impaired migration at 2 µmol/min radical exposure, C16/20 A RhoA mutant podocytes migrate normally. N= 4 viewing areas each group, * p < 0.05 vs. control. (C) Activation of RhoA by lipid radicals is blunted in podocytes bearing C16/20 A mutated RhoA as measure by a “G-LISA” assay (see also Fig. 2). (D) Track direction of C16/20 A RhoA transduced podocytes were followed in random individual cells (colored lines show representatives) for 72 h using an Image J manual cell tracking plug-in. (E-F) Total distance traveled and average velocity have been calculated from the tracks. Podocytes with mutant RhoA display normal migratory parameters. (G) F-actin fibers were visualized and (H-I) anisotropy and fiber orientation were measured similarly to as shown in Fig. 3, in wild type RhoA and C16/20 A RhoA transduced cells using the highest radical concentration. (One-way ANOVA analysis was used for multiple comparisons).