Fig. 3: The killswitch alters the composition, component dynamics and function of the nucleolus.
From: Probing condensate microenvironments with a micropeptide killswitch
a, Schematic of the NuFANCI procedure. b, Protein expression profiles of NuFANCI-isolated nucleoli and their respective input samples. In the left four columns, nucleolar proteins are annotated based on previous proteomics data. N, two clusters of nucleolar proteins. LFQ, label-free quantification. c, The proteomes of GFP-nb–KS-targeted nucleoli and GFP-nb–KSF-to-G-targeted nucleoli compared with the anti-GFP-nanobody control. P values were calculated using one-sided Student’s t-tests. FC, fold change. d, Fixed-cell immunofluorescence images of GFP–NPM1-expressing cells that were untransfected or transfected with GFP-nb, GFP-nb–KS, GFP-nb–2×KS or GFP-nb–KSF-to-G constructs. Scale bars, 5 µm. e, NEPRO fluorescence intensity in nucleoli. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Dunnett’s T3 multiple-comparison test versus the GFP-nb condition. n = 32 (untransfected), 33 (GFP-nb), 36 (GFP-nb–KS), 29 (GFP-nb–2×KS) and 31 (GFP-nb–KSF-to-G) cells from two independent experiments. f, NEPRO fluorescence intensity in nuclei. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Dunnett’s T3 multiple-comparison test versus the GFP-nb condition. The numbers of cells are the same as described in e and are from two independent experiments. g, FRAP analysis of mCherry-signal in HCT-116 cells expressing GFP–NPM1 from the endogenous locus after transfection with the indicated nb constructs and co-transfection with mCherry–SURF6 or mCherry–RPL18. Data are mean ± s.d. n = 10 for all, except for the SURF6 experiment for the TagBFP-only sample (n = 8; asterisk). h, Fixed-cell immunofluorescence images of 5.8S rRNA in GFP–NPM1-expressing cells that were transfected with either the GFP-nb or GFP-nb–2×KS construct. Scale bars, 5 µm. i, Quantification of 5.8S rRNA mean fluorescence intensities in the demixed and remaining portions of nucleoli in GFP-nb–2×KS-expressing cells. Data are mean ± s.d. n represents the number of cells from one experiment. The experiment was repeated twice with similar results. P values were calculated using one-way ANOVA followed by Tukey’s post hoc test. Some elements in the scheamtic in a were created in BioRender. Hnisz, D. (2025) https://BioRender.com/fa5zmne.
