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Fig. 5. In Ect2-depleted cells cleavage furrow formation is impaired, whereas in cells overexpressing the Ect2_1-333 fragment cell abscission fails. (A) Western blots with lysates from a stable tetracycline-inducible myc-Ect2_1-333 cell line, with or without induction with tetracycline for 24 hours. Blots were probed with anti-myc 9E10 antibodies to illustrate induction of the Ect2 fragment and with anti--tubulin antibodies to show equal loading. (B) HeLa S3 cells were treated for 24 hours with either GL2 (control) or Ect2 siRNAs and, in parallel, the inducible myc-Ect2_1-333 cell line was treated for 24 hours with tetracycline. After fixation and permeabilization with paraformaldehyde and Triton X-100, respectively, the myc-Ect2_1-333 expressing cells were stained with anti-myc 9E10 antibodies (red), anti -tubulin antibodies (green) and DAPI (blue) and the siRNA-treated cells were stained with anti-Ect2 antibodies (red), anti--tubulin antibodies (green) and DAPI (blue). Images represent merged deconvolved series of Z stacks through each cell. (C) Live-cell imaging of tetracycline (tet)-induced myc-Ect2_1-333 HeLa S3 cells (upper panel), Ect2-depleted HeLa S3 cells (middle two panels) and HeLa S3 cells treated with GL2 control oligonucleotides (lower panel). Images were taken every 2 minutes and only representative frames are shown. In cells expressing myc-Ect2_1-333, time `0' was attributed to the last frame in which the cell on the right side still showed an interphase appearance. Similarly, in Ect2-depleted and GL2-treated control cells, time `0' was attributed to the last frame in which the relevant cells still showed an interphase appearance. Bars, 10 µm.

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