siRNA-mediated depletion of SEPT7 perturbs actin ring kinetics during WPB exocytosis. (A) WPB fusion was quantified by change in morphology (rod>round) and loss of mCherry signal, whereas actin ring recruitment was assessed by the presence or absence of LifeAct-GFP signal. White asterisk (∗) indicates change in morphology from rod>round. Number sign (#) indicates recruitment of LifeAct GFP ring. HUVECs were co-transfected (2 rounds) with either 300 pM LUC or independent SEPT7-targeted siRNAs and both LifeAct-GFP and p.sel.lum.mCherry. (B) Western blotting was used to determine KD efficiencies of approximately 50% and 82% (averaged from 3 independent experiments). (C) SEPT7 knockdown did not affect vesicle fusion. (D) Quantification of actin ring formation as a percentage of total fusion events. (D-E) Quantification of the duration of the “lifetime” of actin ring (LifeAct-GFP signal) in control and SEPT7 KD cells. ∗∗P < .01, ∗∗∗P < .005. One-way ANOVA with Tukey multiple-comparison test. Frequency distribution (%) of actin ring duration in luciferase siRNA (F) and SEPT7 siRNA-treated cells (84% KD efficiency) (G). Cell numbers for live cell imaging. LUC control, 46 cells; SEPT7, 25 cells (50% KD), and SEPT7, 30 cells (82% KD).