Analysis of nucleoli with high throughput screening technology. (a) Original images of HeLa cells stained with DAPI (blue) and anti-fibrillarin antibodies (red) were acquired with a high throughput imaging system equipped with a widefield optical imaging unit. Cells were incubated with the drugs indicated or the vehicle only (water for cycloheximide, DMSO for all other samples) and processed for indirect immunofluorescence as described in the Methods section. (b-e) Following correction for background, the probe image was analyzed for the presence of nucleoli, which were defined as bright holes that are between 2 and 5 μm2 in size and contain pixel intensities larger than the user-defined threshold. The total number of cells was determined with the DAPI image. It should be noted that the background correction and data analysis were performed in a fully automated fashion without visual inspection of the images. In parts b-e results for three independent experiments are depicted as average + STDEV. (b) For each condition the total number of nucleoli was divided by the number of cells; the results were normalized to DMSO-treated samples. (c) The area stained with anti-fibrillarin antibodies was used to evaluate the effect of different compounds. The threshold was set at 4 μm2; and the percentage of nucleoli below this threshold was determined for each treatment. Several of the drugs increased the percentage of nucleoli that are smaller than 4 μm2. (d, e) The effects of drugs on the pixel intensity (d) and nucleolar area (e) were measured. Statistically significant differences for drugs delivered in DMSO were determined by One-way ANOVA. Data obtained for water and cycloheximide were compared by Student's t-test. Act D, actinomycin D; Meth, methotrexate; Rosc, roscovitine; DRB, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole; CHX, cycloheximide.