Quantitative analysis of B23 and fibrillarin nucleolar localization. The abundance of proteins B23 and fibrillarin in nucleoli is regulated by stress. HeLa cells were grown under normal conditions or heat-shocked for 1 hour at 45.5°C as shown in Figure 1. Nucleolar and nuclear fluorescence was quantified and pixel values/area were calculated. For B23, 21 control cells (38 nucleoli) and 22 heat-shocked cells (36 nucleoli) were measured; for fibrillarin 20 control (28 nucleoli) and 20 heat-treated cells (31 nucleoli) were analyzed. Results obtained for control conditions were defined as 1; significant differences were identified with two-tailed Student's t-test. Raw data were further analyzed in part a-e. (a) Fluorescence intensities were measured for all nucleoli. Individual nucleoli were given the same weight in the analysis, independent of the number of nucleoli in each nucleus. (b) Fluorescence intensities were measured for all nucleoli, average values were calculated for nucleoli if multiple nucleoli were in the same nucleus. Based on these average values, it was determined whether nucleolar fluorescence is different in control and stressed cells. (c) Changes in nucleolar fluorescence/area are shown as histograms. The same binning was applied to control and stressed samples for each protein. (d) Single cell analysis depicting nucleolar fluorescence intensities/area for individual nuclei. Results are shown for individual nuclei; each line represents the measurement for the sum of nucleoli of one nucleus of control (blue lines) or heat-shocked cells (red lines). Data were not normalized. (e) The values obtained for nucleolar fluorescence intensities in part b were used to determine the ratio of nucleolar/nuclear fluorescence intensities. Note that there is no drastic change for B23 upon heat shock, whereas a significant change is observed for fibrillarin.