Figure 4. Arhgap24 knockdown in differentiated podocytes increases membrane ruffling.

(A) Two lentivirally transduced cell lines targeting different portions of the Arhgap24 transcript show marked reduction of Arhgap24 protein compared with that of an irrelevant knockdown (Fluc, 100%; line 451, 15%; line 756, 40%). Results are representative of at least 3 independent experiments. (B) Representative images of the 3 knockdown cell lines after differentiation at 37°C for 7 to 10 days. Kymographs (insets) generated from the hatched box area and time-lapse sequences show that the 2 knockdown cell lines have increased membrane ruffling compared with that of the control (also see Supplemental Videos 3–5). Original magnification, ×400; approximately ×1,000 (insets and time-lapse panels). (C) Quantitation of actin spikes in kymographs shows that both the 451 (n = 19) and 756 (n = 11) Arhgap24 cell lines have significantly greater membrane ruffling activity compared with that of the control knockdown Fluc cell line (n = 18) (*P < 0.00001). The ruffling activity is not significantly different between the 2 knockdown cell lines (P = 0.57). Group differences were analyzed by ANOVA with post-test correction.