Fig. 5: p14^ARF reduces nucleolar NPM1 diffusion in a concentration dependent manner.

A Zoomed in regions from fluorescence microscopy images of live DLD-1^NPM1-G (clone B11) cells, before and after 48 h of doxycycline induced p14^ARF-iRFP expression. Scale bars = 2 µm. B Z-score analysis of NPM1-GFP and p14^ARF-iRFP levels in DLD-1^NPM1-G cells, showing that p14^ARF and NPM1 levels are anti-correlated (statistical significance was assessed by two-sided Mann–Whitney U-test, n = 2272, 122, 54 cells, p-values are shown in the figure) C FRAP curves with fits, as described in Methods, for cells sorted from the DLD-1^NPM1-G population shown in B. The curves on the left are from a cell expressing a high level of nucleolar NPM1 (clone F6; green trace) and a low level of nucleolar p14^ARF (clone F6; blue trace). The curves on the right are from a cell expressing a low level of nucleolar NPM1 (clone G2; green trace) and a high level of nucleolar p14^ARF (clone G2; blue traces). In unsorted DLD-1^NPM1-G cells, D The D_App and E mobility for nucleolar NPM1-GFP and p14^ARF-iRFP (small green and blue transparent markers, respectively, n = 45 cells) are reduced as nucleolar p14^ARF-iRFP levels increase. Reductions also occur as the duration of p14^ARF-iRFP expression is extended (large opaque markers; scatter points represent the mean and error bars represent the standard deviation, where n = 20 cells). F A schematic describing the correlated reductions in p14^ARF and NPM1 dynamics and their assembly into large molecular weight complexes within the granular component (GC) of the nucleolus.