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. 2022 Jul 21;18(10):1104–1114. doi: 10.1038/s41589-022-01062-y

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a, Schematic representation of the molecular interactions of NPM1 within the nucleolus and the nucleoplasm. b, Comparison of the self-association property (in log₂ scale, y axis) of the indicated SNAP-tagged phosphomutants of NPM1 (x axis) normalized to that of SNAP-tagged NPM1 WT. This was measured by assessing the amount of HeLa cell (native) NPM1 associated with heterologously overexpressed SNAP-tagged NPM1 variants following immunoprecipitation (IP) with SNAP-tag as bait. Points represent data from three independent trials and the bar represents the mean value. Significance for each phosphomutant pair was calculated using Student’s t-test (two sided) and is represented by *P < 0.05, **P < 0.01 and ***P < 0.001. c, Comparison of the amount of 28S rRNA (in log₂ scale, y axis) associated with the indicated SNAP-tagged phosphomutants of NPM1 (x axis) normalized to that of SNAP-tagged NPM1 WT following IP with SNAP-tag as bait. Points represent data from three independent trial and the bar represents the mean values. Significance for each phosphomutant pair was calculated using Student’s t-test (two sided) and is represented by *P < 0.05, **P < 0.01 and ***P < 0.001. d, Comparison of the relative amounts of NPM1-interacting proteins (in log₂ scale, y axis; 44 proteins were classified as NPM1 interactors, including both ribosomal and non-ribosomal proteins) associated with the indicated SNAP-tagged phosphomutants of NPM1 (x axis) in comparison to those associated with SNAP-tagged NPM1 WT following IP with SNAP-tag as bait. Significance for each phosphomutant pair was calculated using Student’s t-test and is represented by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. The box plots display the median and the IQR, with the upper whiskers extending to the largest value ≤1.5 × IQR from the 75th percentile and the lower whiskers extending to the smallest values ≤1.5 × IQR from the 25th percentile. e, Heatmap showing normalized relative effect sizes of partition coefficient values (K) and amount of native NPM1 (reflects NPM1 self-association), rRNA and protein interactors of NPM1 associated with the indicated phosphomimetic mutants (x axis). Significance levels were obtained with Student’s t-test (two sided) and are represented by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. f, Schematic representation of the working model of the impact of phosphorylation on NPM1’s propensity to form a biomolecular condensate. Pink dots represent phosphosites, gray circles indicate NPM1, diamond shapes represent r-proteins, and the stem-loop schematic represents rRNA.

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