Fig. 5. Supervillin knockdown increases the rate of cell spreading. (A) Immunoblots of endogenous SV, MHC IIA, MHC IIB, long and short isoforms of MLCK (L-MLCK and S-MLCK), and ERK1/2, in lysates from cells treated with transfection reagent alone (lanes 1; Mock), dsRNAs 1680 (hSV1) or 2026 (hSV2) that target SV (lanes 2, 3), or control dsRNAs (lanes 4, Con). (B) Histogram showing the rates of area change for populations of A549 cells treated with control (white) or hSV1 (black) dsRNAs spreading on 10 µg/ml fibronectin. Spreading velocities in the bimodal distributions typical of unsynchronized cells (Dubin-Thaler et al., 2004) appear to be displaced, but the difference between the means (36.2±5.7 µm²/minute for controls; 51.7±11.3 µm²/minute for hSV1) is not statistically significant for the numbers of living cells (n=16; n=17) assayed. Movies 1 and 2 in the supplementary material show cell morphologies during linear spreading for average control- and hSV1-treated cells. (C) Percentage of spread A549 cells after plating onto 10 µg/ml fibronectin at 15, 30, 45, 60 or 90 minutes. Spread cells were defined as in Fig. 1; the average diameter of a rounded A549 cell was taken as 18.0±0.8 µm (means±s.e.m., n=18). Cells were mock-transfected, or were treated with hSV1, hSV2 or control dsRNA. Means±s.e.m. of 200 cells counted per experiment, n=5. (D) Percentage of spread A549 cells plated on 10 µg/ml fibronectin for 30 minutes. Means±s.e.m.; n=5; ^*P<0.05 for each SV dsRNA versus both mock and control dsRNA treatments.