Supplemental Materials

This article contains the following supporting material:

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• Supplementary figure S1 - Progression through metaphase is delayed in myosin VI KD cells. Mitotic phase indexes of mock transfected cells and myosin VI KD cells using myosin VI smartpool siRNA primer are shown in (A). Control and KD cells were fixed and stained with anti-tubulin antibodies and Hoechst dye and scored for mitotic phase on the basis of chromosome configuration and distribution and spindle morphology. All values are means from at least 5 different experiments scoring 3000 control cells, 1500 KD cells for primer 1 and 3000 KD cells for primer 2. The number of KD cells in metaphase was significantly higher as compared to control cells. (B) Control and myosin VI progression through mitosis were monitored using DIC time lapse microscopy. Pictures were taken every 60 s and the time was measured from the first appearance of chromosomes at the metaphase plate until chromosomes started to move apart in anaphase. Whereas in control cells this process took less then 30 min, KD cells took more then 30 or even 60 min to progress into anaphase.
• Supplementary figure S2 - Marker proteins of the Golgi complex and the trans-Golgi network are not colocalising with myosin VI in the midbody region. To analyse whether marker proteins of the Golgi complex or the trans-Golgi network (TGN) and post-Golgi membrane trafficking colocalise with myosin VI in the midbody region, stable MDCK cells expressing GFP-myosin VI (a, d and g) were fixed and stained with antibodies to the Golgi matrix protein GM130 (b), the TGN/post-Golgi SNARE vti1a (e) or the TGN/Golgi SNARE syntaxin 6 (h). The merge images together with the DNA stain in blue are shown in (c, f and i). During cytokinesis none of the Golgi or post-Golgi membrane trafficking marker proteins was recruited to the midbody region, where myosin VI was present.
• Supplementary figure 3 - Myosin II and actin are present in the intercellular bridge in MDCK during cytokinesis. To confirm that actin and myosin II are present in the intercellular bridge during late cytokinesis in MDCK cells, stable MDCK cells expressing GFP-myosin VI (a, d and g) were fixed and stained with antibodies to myosin II (e and h) or for F-actin with Rhodamine phalloidin (b). The merge images together with the DNA stain in blue are shown in (c, f and i).
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