The perturbation of ring-like F-actin structure formation by chemical inhibitors and real-time imaging analyses.

<p>(A) The method to measure the apparent intensity of ring-like F-actin structure. A cell is classified into four regions named circle 1, 2, 3 and 4. Circle 1 includes the region of spindle-chromosome complex; circle 2 includes the region of ring-like F-actin structure just around the spindle, excluding the region inside circle 1; circle 3 includes the region of all cytoplasmic F-actin just inside the cortex, excluding the region inside circle 2; circle 4 includes the region of the whole cell body, excluding the region inside circle 3. The formulae to calculate apparent intensity are displayed here. (B) Representative images of a HeLa cell treated with DMSO and MG132. Cells were double blocked and released. MG132 was added at time point -5′, and DMSO was added at 0′. We acquired each image every 1 minute since -10', and the total time was 1 hour. Cells were transfected with GFP-tubulin (green) and mCherry-UtroCH (red) to label microtubules and F-actin, respectively. Scale bar, 5 µm. (C) Representative images of HeLa cells treated with DMSO, BI2536, Blebbistatin, Reversine and Lat B. We used the same method as (B). The influence of chemical treatment on the ring-like structure and spindle position can be observed and measured from the supplemental movies. Scale bar, 5 µm. (D) The apparent intensity of the ring-like structure in each cell measured from the living cell imaging. The curve of circle 3 represents the photo-bleaching along the time. We chose the apparent intensity of circle 3 as the normalization factor. We also chose the apparent intensity of circle 2 as the apprent intensity of ring-like F-actin structure.