a Actinin and Mechanobiology Figure 1. Expression, localization, and suppression of a-actinin isoforms in glioma cells. (A) Localization of a-actinin-1 and a-actinin-4 in U-373 MG cells cultured on collagen-coated glass. Solid and open arrowheads mark the localization of individual a-actinin isoforms at ruffles and along actin stress fibers, respectively. Scale Bar = 10 mm. (B) Measurement of siRNA efficacy by Western Blot of U-373 MG cells transfected with either scrambled control siRNA (siCTL) or siRNA directed against a-actinin-1 (siACTN1) or a-actinin-4 (siACTN4) (*p,0.05). (C) Subcellular localization of a- actinin-1 and a-actinin-4 following siRNA treatment. Arrowheads depict the redistribution of a-actinin-4 to FAs when a-actinin-1 is suppressed. Scale Bar = 20 mm. doi:10.1371/journal.pone.0008427.g001 of each isoform with isoform-specific siRNA sequences (siACTN1 and siACTN4 respectively) and compared the results of each treatment on expression and subcellular localization to each other and to a scrambled siRNA control sequence (siCTL) (Figure 1B). Individual isoform-specific siRNA sequences and transfection conditions were optimized to obtain ,55% depletion of a-actinin- 1 and ,60% depletion of a-actinin-4 by immunoblot, comparable to levels of suppression achieved in previous studies with minimal off- target effects [18]. Consistent with this incomplete knockdown, a- actinin-1 and a-actinin-4- depleted cells still stained weakly positive for a-actinin-1 and a-actinin-4 respectively (Figure 1C, Figure S1). Although depletion of one isoform did not cause compensatory changes in expression of the other isoform, a-actinin-1 suppression led to partial redistribution of a-actinin-4 to FAs (arrows). This was also confirmed by co-staining cells with isoform-specific a-actinin antibodies and an antibody against the phosphorylated Tyrosine-397 of focal adhesion kinase (pY397FAK), a marker of mature focal adhesions (Figure S1). a-Actinin Isoforms Contribute to Glioma Cell Motility We next quantified the effect of depleting each a-actinin isoform on cell motility in vitro by tracking the random migration of control and knockdown cells using phase-contrast time-lapse imaging (Figure 2, Movies S1, S2, S3). The contributions of individual a- actinin isoforms to glioma motility were quantified by calculating Figure 2. Contributions of a-actinin isoforms to glioma cell motility. Effect of a-actinin depletion on mean speeds (mean6SEM, *p,0.001) of cells tracked over 6 hours. doi:10.1371/journal.pone.0008427.g002 PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8427