Visualization of inducible <i>trans</i> association in 3D in whole cells.

<p><u>A, B</u> – spot counting in fixed cells. <u>A. Representative examples of cells with 1 spot and 2 spots.</u> Cells with one round spot were classified as “1 spot – cells”. Cells with two discernable spots, no matter how close to one another, were classified as “2 spot – cells”. <u>B. Quantitation of the microscopic analysis.</u> Percentages of cells with 1 spot, 2 spots and >2 spots (3 or 4 spots) are plotted for a culture without β-estradiol (light grey bars) and a culture with β-estradiol (dark grey bars). Three independent cultures were analyzed; 500 cells per each sample were scored. <u>C–H. Spot localization analysis in live cells. C. A cartoon showing z-planes.</u> The z-plane with the brightest spot is highlighted. <u>D. Examples of z-planes with the brightest spots.</u> The spots were computationally enhanced for publication purposes. <u>E. Measurements.</u> The definition of the nuclear periphery was based on the perinuclear localization of Nup49-GFP, which manifested itself as a close curve enveloping the nucleus with an average 250 nm thickness (green oval); the true nuclear perimeter was assumed to be centered within this band. In the z-plane with the brightest spot, three parameters were measured: the shortest distance from the center of the spot to the estimated true nuclear periphery (L), long axis of the nucleus (d1) and short axis of the nucleus (d2). The average diameter was calculated by taking the arithmetic mean of the long and short axis. Each spot distance to the nuclear periphery was then normalized to the corresponding average diameter, and divided by two to bring values to the 0–1 scale, where 0 is the periphery and 1 is the center. <u>F. Analysis of nuclear diameters.</u> Left – histogram of d1/d2, where d1 is the longest nuclear axis and d2 is the shortest nuclear axis. Relationship between d1 and d2 is a measure of roundness of nuclei. Right – distribution of average diameters, which were calculated as arithmetic mean of the long and short axis for all categories of cells combined. <u>G. Equal area zones.</u> Normalized distances (L′) were binned into three zones with equal areas. The borders of the zones were defined as follows: 0.184, 0.422, 1 (where 0 is nuclear periphery and 1 is nuclear center). <u>H. Percent of cells with spots in each of the three zones.</u> Four categories of cells were analyzed: 1 spot cells from the culture without β-estradiol (light grey bars); 1 spot cells from the culture with β-estradiol (dark grey bars); 2 spot cells from the culture without β-estradiol (light brown bars); 2 spot cells from the culture with β-estradiol (dark brown bars). Dotted line illustrates the hypothetical scenario on which spots are randomly distributed between the three equal area zones.