(A–D) siRNA-based depletion of muskelin leads to morphological alterations

(A) Reduced attachment of A549 cells with stable expression of muskelin-shRNA clone 2 (cl2) and clone 3 (cl3) cells to the TSP-1 C-terminal protein T3GCT. Adhesion assays were performed for 1 h under serum-free conditions as previously described (). (B and C) Cell perimeter measurements for control and muskelin-depleted A549 (B) or C2C12 (C) cells adherent on 50 nM FN for 1 h. (B) A549 lines were also compared after 48 h expression of GFP or GFP-MK. Only GFP-MK restored normal morphology. In each graph, each column represents the mean of three independent experiments. Error bars indicate SEM. *, P ≤ 0.0001. (D) Representative F-actin organization in control and muskelin-depleted C2C12 cells under the same experimental conditions as in C. Bar, 10 μm. (E) Activity of muskelin mutants in restoring the morphology of muskelin-depleted A549 clone 3 cells. GFP, GFP-MK, or the indicated mutants were expressed in clone 3 cells for 48 h, the cells adhered to 50 nM FN for 1 h, and cell perimeter lengths of GFP-positive cells were measured. Each column represents the mean of three independent experiments. Error bars indicate SEM. *, P ≤ 0.00001. At least 500 cells were scored per condition. (F) RanBP9 knockdown results in similar morphological changes in FN-adherent A549 cells. Where indicated, cells were treated with 0.75 μg/ml doxycycline for 36 h. Each column represents the mean from >50 cells. Error bars indicate SEM. *, P < 0.0001. Insets show representative merged cell images of F-actin (green), DAPI (blue), and, where induced, shRNA transcript expression (red; RFP). Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Novel role of the muskelin–RanBP9 complex as a nucleocytoplasmic mediator of cell morphology regulation"</p><p></p><p>The Journal of Cell Biology 2008;182(4):727-739.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518711.</p><p>