Figure 3.
TAO1 is required to maintain dynamic microtubules during mitosis. (a) Immunofluorescence microscopy images showing the presence of ‘long-lived and cold-stable’ microtubules in TAO1-depleted cells (yellow arrows). HeLa cells were transfected with the indicated siRNA, cold treated for 20 min and immunostained with acetylated tubulin antibodies (green) and DAPI for DNA staining (blue). Scale bar, 5 μm. (b) Graph showing percentage of interphase cells displaying long-lived and cold-stable microtubules following siRNA and cold treatment as described in (a). n = number of cells from three independent experiments. (c) Deconvolved immunofluorescence images illustrating the reduction in EB1 comets following TAO1 siRNA treatment. HeLa cells were transfected with the indicated siRNA and immunostained with antibodies against α-tubulin (blue), EB1 (green) and CREST antisera (red). Scale bar, 5 μm. (d) Still images from time-lapse movies illustrating the reduction in comet density in HeLaEB3-Tomato cells following TAO1-St1 or Control-St siRNA treatment. HeLaEB3-Tomato cells were treated with siRNA as indicated and imaged during interphase or mitosis as indicated. Scale bar, 20 μm (interphase) and 5 μm (mitosis) images; inset 5 μm. (e) Graph of normalized average density of EB3 comets in HeLaEB3-Tomato interphase cells counted as described in methods. n = 6 cells for each condition. Error bars are s.e.m. values across three experimental repeats. p-Values representing significance level were derived using the proportion test. Asterisks (*) indicate significant difference.