Development of inducible fascin-knockdown SW480 cells. (A) Immunoblot for fascin expression in human colon carcinoma cell lines. C2C12 skeletal myoblasts were used as a normal cell control. (B) Development of SW480 cells expressing TetR (PaA) and inducible fascin-knockdown clones F11 and F12. Doxycycline was applied at 1 μg/ml for 72 h. Graph shows normalized fascin levels (±SD) by scanning densitometry from triplicate experiments. (C) Time and concentration dependence of fascin knockdown in IKD-F11 cells. (D) Indirect immunofluorescent staining for fascin, in IKD-F11 cells maintained in the absence or presence of 0.5 μg/ml doxycycline for 72 h. Bar, 20 μm. Inset, detail of fascin-containing protrusions and associated filopodia. Bar, 10 μm. (E) Persistence of fascin knockdown. IKD-F11 cells were left untreated as controls or treated with the indicated concentrations of doxycycline for 48 h (shaded doxycycline panel), and then they were cultured in the absence of doxycycline for 72 h (unshaded, no doxycycline). The total experimental time was 120 h. Replicate cultures were lysed at the indicated timepoints and the levels of fascin protein examined by immunoblot during and after doxycycline treatment. Fascin knockdown in samples that had received doxycycline persisted for 72 h. Molecular mass markers are in kilodaltons.