The SEPT9 isoforms are not all functionally equivalent. (A and B) Stable cell lines inducibly expressing siRNA-resistant Flag-SEPT9_i3 (A) or Flag-SEPT9_i4 (B) were treated with increasing amounts of doxycycline, and expression was assayed by Western blotting. (C) The parent and Flag-SEPT9_i3 cell lines were treated with control or SEPT9 siRNA in the absence of doxycycline, and SEPT9 levels were assayed by Western blotting. Note that SEPT9_i4 disappears upon treatment with SEPT9 siRNA, demonstrating efficient KD. Also note the higher SEPT9 (_i1-3) levels in the Flag-SEPT9_i3 stable line upon SEPT9 depletion, when compared with the parent line. (D) The parent and Flag-SEPT9_i4 cell lines were treated as in C. Numbers to the right of the blots represent molecular mass standards in kilodaltons. (E) The parent, Flag-SEPT9_i3, and Flag-SEPT9_i4 cell lines were treated with control or SEPT9 siRNA and assayed for defects in cytokinesis by immunofluorescence microscopy. Unresolved cytokinesis refers to cells exhibiting midbody attachment or multinucleation. Data are represented as mean ± SEM (error bars; n ≥ 300 cells from three or more independent experiments). *, P < 0.05; **, P < 0.005.