Figure 7.
ECT2-BRCTTK Supports Cytokinesis
(A) Immunoblot analysis of protein lysates from monoclonal cell lines stably expressing the indicated transgenes. Lysates were prepared 48 hr after transfection with NTC (−) or ECT2 siRNA (+). Endogenous ECT2 protein and transgenic AcFL-ECT2r are indicated by open and filled arrowheads, respectively. All stable cell lines express the transgenes in more than 95% of cells.
(B) IF analysis of indicated cell lines. Cells were transfected with ECT2 siRNA. Multi-nucleation levels were analyzed 48 hr after transfection (n > 300 cells each from three independent experiments). The scale bars in this panel and the following panels represent 10 μm.
(C) Representative images showing cytokinetic phenotypes for indicated cell lines after depletion of endogenous ECT2. Cells were imaged with bright field microscopy from 24 hr after siRNA transfection onward. Time point t = 0 min was set to the metaphase-to-anaphase transition.
(D) Quantification of cytokinetic phenotype from recordings in (C). Mono-nucleated cells undergoing cell division were scored from 24 to 48 hr post-transfection (n > 300 each, bars represent mean values of three independent experiments).
(E) Quantification of cytokinetic phenotype of cells stably expressing ECT2-BRCTTK and transfected with different siRNA combinations as indicated in the graph. Cells were imaged with bright field microscopy from 24 hr after transfection onward. Mono-nucleated cells undergoing cell division were scored from 24 to 60 hr post-transfection (n > 100 cells each, bars represent mean values of three independent experiments).
(F) IF analysis and quantification of RhoA and anillin localization in anaphase cells expressing the AcFL tag, ECT2-WT, or ECT2-BRCTTK. Cells were transfected with ECT2 siRNA and synchronized by thymidine release protocol. Cells were fixed and stained 36 hr after transfection. The fluorescent intensity profile along the cell membrane for RhoA and anillin in anaphase cells is plotted as the ratio of the mean signal intensity at the cell periphery and the cytoplasm against the measured length (n = 15 cells, lines represent mean values).
See also Figure S6.