ECM or altering processing of microenvironmental mechanical cues. Since our level of actinin suppression is partial, some of our observations may be consequences of altered myosin or vinculin expression. However, it is also possible that modulation of a- actinin expression in vivo may trigger compensatory changes in the expression and function of other FA proteins, as we observe in culture. Testing this hypothesis will require asking whether normalizing a-actinin overexpression can reduce invasive behavior in vivo. Given the importance of NMMII to navigating micro- structural barriers in 3D ECMs, it will also be important to revisit these behaviors in the context of 3D ECMs that present more complex combinations of mechanical and topological cues. Supporting Information Figure S1 Immunofluorescence co-localization of pY397FAK with (A) a-actinin-1 and (B) a-actinin-4 following siRNA-mediated suppression of each a-actinin isoform in U-373 MG cells.In both (A) and (B), the left, middle, and right columns show results for control, a-actinin-1 and a-actinin-4-directed siRNAs, respectively. In each case, the top, middle, and bottom rows show immunolocalization of the relevant a-actinin isoform (green), pY397FAK (red), and the merged signal, respectively. Scale Bar = 20 mm. Found at: doi:10.1371/journal.pone.0008427.s001 (6.13 MB TIF) Figure S2 Immunofluorescence localization of vinculin (red) in control cells (siCTL) (left column), a-actinin-1-depleted cells (siACTN1) (middle column), and a-actinin-4-depleted cells (siACTN4). Five representative images of each category are shown. Scale Bar = 20 mm. Found at: doi:10.1371/journal.pone.0008427.s002 (7.44 MB TIF) Figure S3 Immunofluorescence localization of pY397FAK in control cells (siCTL) (left column) a-actinin-1-depleted cells (siACTN1) (middle column) and a-actinin-4-depleted cells (siACTN4) (right column). Cells have been co-stained for pY397FAK (red) and F-actin (green). Scale Bar = 20 mm. Found at: doi:10.1371/journal.pone.0008427.s003 (7.36 MB TIF) Figure S4 Effect of nonmuscle myosin II pathway inhibitors on phosphorylated MLC (pMLC) levels in U-373 MG cells by Western Blot. From left to right, the lanes represent lysates from untreated controls (CTL), cells treated with 10 mM ML7 (ML7), cells treated with 10 mM Y27632 (Y27), and cells treated with 10 mM blebbistatin (Blebb). The bottom bands are corresponding GAPDH loading controls.M ML7 (ML7), cells treated with 10 mM Y27632 (Y27), and cells treated with 10 mM blebbistatin (Bleb). The bottom bands are corresponding GAPDH loading controls. Found at: doi:10.1371/journal.pone.0008427.s004 (1.10 MB TIF) Figure S5 Effect of a-actinin suppression on pMLC levels by Western Blot. From left to right, the lanes represent lysates from cells treated with control siRNA (siCTL), siRNA against a-actinin- 1 (siACTN1), and siRNA against a-actinin-4 (siACTN4). The bottom bands are corresponding GAPDH loading controls. Found at: doi:10.1371/journal.pone.0008427.s005 (1.03 MB TIF) Figure S6 Time course of effect of a-actinin-1 suppression on expression of vinculin and NMMII by Western Blot. The leftmost lane represents lysates from cells treated with control siRNA for 8 hours (siCTL). Subsequent lanes represent cells treated with siRNA against a-actinin-1 (siACTN1) for 8, 16, and 24 hours, respectively. Blots reveal a time-dependent suppression in NMMII expression (top bands) and a gradual increase in vinculin expression (middle bands). The bottom bands represent the GAPDH loading controls. a Actinin and Mechanobiology Found at: doi:10.1371/journal.pone.0008427.s006 (1.18 MB TIF) Figure S7 Traction maps exerted by siCTL, siACTN1, and siACTN4 cells on 2 kPa gels. Traction fields have been computed using Fourier Transform Traction Cytometry. Arrows show the direction and relative magnitude of traction forces. Color code shows the magnitude of traction forces in Pa. Note the differences in scales. Found at: doi:10.1371/journal.pone.0008427.s007 (6.66 MB TIF) Movie S1 Phase contrast imaging of U373-MG cells transfected with control siRNA (siCTL) migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s008 (9.05 MB AVI) Movie S2 Phase contrast imaging of U373-MG cells transfected with a-actinin-1 siRNA (siACTN1) migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s009 (9.05 MB AVI) Movie S3 Phase contrast imaging of U373-MG cells transfected with a-actinin-4 siRNA (siACTN4) migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s010 (9.05 MB AVI) Movie S4 Phase contrast imaging of U373-MG cells transfected with control siRNA (siCTL) and exposed to 10 mM ML7, migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s011 (10.14 MB AVI) Movie S5 Phase contrast imaging of U373-MG cells transfected with control siRNA (siCTL) and exposed to 10 mM Y27632, migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s012 (10.14 MB AVI) Movie S6 Phase contrast imaging of U373-MG cells transfected with a-actinin-1 siRNA (siACTN1) and exposed to 10 mM ML7, migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s013 (10.14 MB AVI) Movie S7 Phase contrast imaging of U373-MG cells transfected with a-actinin-1 siRNA (siACTN1) and exposed to 10 mM Y27632, migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s014 (10.14 MB AVI) Movie S8 Phase contrast imaging of U373-MG cells transfected with a-actinin-4 siRNA (siACTN4) and exposed to 10 mM ML7, migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s015 (10.14 MB AVI) Movie S9 Phase contrast imaging of U373-MG cells transfected with a-actinin-4 siRNA (siACTN4) and exposed to 10 mM Y27632, migrating on collagen-coated tissue culture plastic over a period of at least 6 hours. Frames were acquired every 15 min. Found at: doi:10.1371/journal.pone.0008427.s016 (10.14 MB AVI) PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8427