(A,B) gDrp1-U2OS cells were transiently transfected with mitochondrial matrix marker, mito-BFP, and actin filament marker, mApple-F-tractin. Cells were treated with 0, 0.1, 0.2, 1 and 2 μM LatA (or DMSO) for 15 min before imaging. At 60 s after starting imaging, cells were treated with 4 μM ionomycin (in the presence of the appropriate concentration of LatA) and imaged for 9 min. GFP-Drp1 signals over cytosolic background (background subtract, ImageJ) were measured per whole cell; actin filament signals were quantified from two or three ROIs per cell (approximately 3 × 3 μm), in which no stress fibers or cell edges were included. Error bars, standard deviation for A and S.E.M. for B. (C) Time course of ionomycin-induced changes in Drp1 oligomer and actin filaments after INF2 siRNA treatment (INF2 siRNA). 11 control cells (sc siRNA), six INF2 siRNA. Error bars, S.E.M. (D) Time course of ionomycin-induced changes in Drp1 oligomer and actin filaments after myosin IIA siRNA treatment. N = 11 control cells (sc siRNA), 12 myosin IIA siRNA. * denotes total Drp1 puncta or polymerized actin (as indicated for individual curves). Error bars, S.E.M for C and D.