Suppressing RSF assembly reduces the quantity of active myosin II but increases lamellar actin retrograde flow rate. (A) Immunofluorescence images of pMLC and fluorescent phalloidin staining of F-actin are shown for WT, Dia Inh, and Atn-1 KD U2OS cells. Bar, 20 µm. (B) Western blots showing MLC and pMLC for each condition in relation to a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. (C) Densitometry analysis of Western blots showing the relative levels of pMLC, MLC, and the ratio of pMLC to MLC for each condition, normalized to WT cells. There is no significant difference in the ratio of MLC to pMLC for each condition. The bar plot shows the mean ratio relative to WT cells (error bars indicate SEM; n = 6 for each condition). (D) A series of images of GFP-actin expressed in a protruding Atn-1 KD cell over time. Time is indicated in minutes/seconds. Bar, 10 µm. See Video 1. (E) Magnified images of the boxed region indicated at time 0 in D, illustrating the reorientation of a transverse arc to align in the direction of retrograde flow (red arrowheads). Bar, 2 µm. (F) Actin flow vectors are overlaid on the GFP-actin image from E at 4 min and 30 s. The white arrow represents 15 nm/s. Bar, 10 µm. (G) A box plot of the lamellar retrograde flow speed for each condition described (open circle = mean; box = 25th, 50th, and 75th percentile; whiskers = 5th and 95th percentile; n > 150 flow vectors from multiple regions in three to five cells for each condition; ***, P < 0.001 with respect to WT).