Fig. 3.

Lipid peroxyl radicals induce cytoskeletal rearrangements in podocytes and increase RhoA activation. (A) Upper panels: Representative images of untreated podocytes and podocytes incubated with AAPH to generate alkyl radicals (R^•). After incubation (4 h), cells were stained for F-actin as described in Materials and methods. F-actin orientation was observed under a fluorescent microscope (10 pictures/group at random viewing areas). Podocytes exposed to lipid radicals display cortical rearrangement and loss of F-actin transversal fibers. Lower panels: representative greyscale images of control and R^• treated podocytes for anisotropy and orientation analysis, showing the anisotropy vectors in red (Image J “FibrilTool” application). Yellow arrows show examples of actin-rich centers in podocytes exposed to lipid peroxidation. (B) Anisotropy analysis of control and lipid radical treated podocytes confirms significant loss of transversal fibers. (C) Orientation analysis of F-actin fibers, showing significant reorientation in podocytes exposed to lipid peroxidation. Orientation angles from − 90 to + 90 degrees were grouped into 20 degree intervals. Duplicate experiments in six well plates, n = at least 30 cells analyzed per group (3 cells each picture, 10 pictures each group), *p < 0.05 vs. control. (D) Lipid radicals generated from AAPH activate RhoA in a dose-dependent manner. RhoA activation was measured using an active (GTP-bound) RhoA specific “G-LISA” assay. Duplicate experiments, n = 4 per group, *p < 0.05 vs. control (one-way ANOVA for multiple comparisons).