Figure 8: RhoA activity and podocyte function in Myo9A^R701X kidneys and Myo9A^KD podocytes.

A-C) Active RhoA (GTP-RhoA) is increased in Myo9A ^R701X mutants vs. wild type: >2-fold in whole kidneys (A), ~60% in isolated glomeruli (B), >2-fold in primary podocytes (C), Western blots representative of n=3–4 independent experiments, lysates pooled from 3 mice each, quantitation (mean±SD), ANOVA (A) or unpaired t-test (B and C), * P<0.05, ** P<0.001. D) Podocyte migration assay (wound assay)⁵⁷: quantification of the migration area shows that Myo9A^R701X/+ (Het) podocyte migration is decreased; pre-incubation with Y26732 partially abrogates the migration defect, *** P<0.0005 (ANOVA),**** P<0.0001 (unpaired t-test). E) Podocyte attachment assay shows decreased attachment of Myo9A^R701X/+ (Het) podocytes; pre-incubation with Y26732 corrects the attachment defect, *** P<0.0005 (ANOVA). D-E) Data are mean±SD, n=3–4 independent experiments, n=3 mice/genotype/experiment. F) Podocyte migration assay: siRNA-mediated Myo9A knockdown (Myo9A^KD) decreases immortalized mouse podocytes migration (* P<0.05, ANOVA, ** P<0.01, unpaired t-test), whereas migration of non-targeting siRNA-treated podocytes (N-T) is not different from normal podocytes (Wt). G) Attachment assay: Myo9A^KD decreases immortalized mouse podocytes attachment, as compared to podocytes treated with non-targeting siRNA (N-T) and untreated Wt podocytes. Myo9A^KD data are mean±SD, n≥3 independent experiments. * indicates P<0.05, ** P<0.01, ns indicates no significant difference.