Fig. 4: The killswitch alters the compositions and functions of oncoprotein condensates.
From: Probing condensate microenvironments with a micropeptide killswitch
a, Live-cell fluorescence microscopy images of U2OS cells expressing the indicated fusion oncoproteins tagged with GFP. GFP–EWS::FLI was imaged in MCF7 cells. One nucleus is shown in each image. The cells were co-transfected with a GFP-nb, GFP-nb–KS or GFP-nb–KSF-to-G construct. Scale bars, 5 µm. b, FRAP analysis of GFP-fusion-oncoprotein condensates. Data are mean ± s.d. n values are as described in c. c, Quantification of GFP intensity in the bleached condensates. n = 10 cells, except for TAZ::CAMTA1 (n = 14, 19 and 19 cells) and PAX::FOXO1 (n = 11, 14 and 15 cells) for GFP-nb, GFP-nb–KS and GFP-nb–KSF-to-G, respectively, from two independent experiments. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Tukey’s post hoc test. d, Fixed-cell immunofluorescence images of RNAPII in cells expressing GFP–BRD4::NUT, GFP–BRD4::NUT–KS, or GFP–BRD4::NUT–KSF-to-G. Scale bars, 5 µm. IF, immunofluorescence; r, Pearson correlation coefficient. e, Differential gene expression analyses. Known BRD4::NUT targets are coloured. P values were determined using the Benjamini–Hochberg method. f, Schematic of the generation of the GFP–NUP98::KDM5A-expressing AML model. g, Growth curves of primary mouse AML cells that were transduced with nanobody constructs co-expressing mCherry. The cumulative cell number was recorded after sorting mCherry-positive cells. Data are mean ± s.d. n = 3 biologically independent experiments. h, Fluorescence microscopy images of GFP–NUP98::KDM5A condensates in primary mouse AML cells expressing doxycycline (DOX)-inducible nb–KSF-to-A or nb–KS. The experiments were repeated independently three times with similar results. Scale bar, 5 µm. i, The number of condensates and the mean GFP intensity in the AML cells expressing the indicated nanobody constructs. Data are mean ± s.d. P values were calculated using unpaired two-tailed t-tests. n = 100 cells in all cases examined over three biologically independent experiments. PI, proteasome inhibitors. j, Representative images showing proteasome inhibition partially rescues the GFP–NUP98::KDM5A level in cells expressing nb–KS. The experiments were repeated independently three times with similar results. Scale bar, 5 µm. k, Representative FRAP images of GFP–NUP98::KDM5A nuclear condensates in HEK293T cells. The experiments were repeated independently three times with similar results. Scale bar, 1 µm. Some of the elements in f were created in BioRender. Hnisz, D. (2025) https://BioRender.com/fa5zmne.
