Figure 1.

Membrane Association of ECT2 Is Essential for Cytokinesis

(A) Domain organization of AcFL-tagged and siRNA-resistant full-length ECT2-WT protein (reference sequence NCBI: NP_001245245) and ECT2-C1B protein.

(B) Immunoblot analysis of protein lysates from cells stably expressing the indicated transgenes. Lysates were prepared 48 hr after transfection with NTC (−) or ECT2 siRNA (+). The transgene is expressed in more than 95% of cells.

(C) Live-cell imaging of ECT2-C1B proteins. Stable cell lines expressing ECT2-C1B or ECT2-C1B^Q27G (white) were transiently transfected with H2B-mCherry (red). Cells were imaged 48 hr after transfection and treated with TPA at t = 0 min. Scale bars in this and the following panels represent 10 μm.

(D) Immunofluorescence (IF) analysis of ECT2-C1B cell lines. TPA or DMSO was added 6 hr after transfection with ECT2 siRNA. Multi-nucleation was analyzed 48 hr after transfection (n > 300 cells each from three independent experiments).

(E) Live-cell imaging of indicated cell lines. Cells were transfected with ECT2 siRNA and treated with TPA as in (D). Cells were imaged from 24 hr after transfection. Time point t = 0 min was set to the metaphase-to-anaphase transition.

(F) Quantification of cytokinetic phenotypes using live-cell imaging as described in (E). Mono-nucleated cells undergoing cell division were scored from 24 to 72 hr after transfection (n > 100 each, bars represent mean ± SD of three independent experiments).

See also Figure S1.