Extended Data Fig. 10: NUP98::DDX10 mutagenesis and 1,6-hexandiol treatment on cells expressing GFP-NUP98::DDX10 condensates targeted with the killswitch.
From: Probing condensate microenvironments with a micropeptide killswitch
a. Live cell fluorescence microscopy images of U2OS cells expressing GFP-NUP98::DDX10 constructs each co-transfected with mCherry-CRM1. Scale bar: 5 µm. b. FRAP of GFP-NUP98::DDX10 (top), and of mCherry-CRM1 (bottom) in GFP-NUP98::DDX10 condensates in U2OS cells exemplified in a. Data are mean ± s.d. n = 10 (except 9 for KSF-to-G) in all cases. c. (top) Quantification of GFP intensity in the bleached condensates. (bottom) Control quantification of mCherry intensity in the bleached condensates. Data are mean ± s.d. P-values are from Tukey’s post-hoc test after one-way ANOVA. n = 10 (except 9 for KSF-to-G) cells in all cases from two biologically independent experiments. d. Schematic representation of the NUP98::DDX10 control and mutagenesis constructs. The schematics are in scale to the actual amino acid positions. e. Live cell fluorescence microscopy images of U2OS cells expressing the GFP-NUP98::DDX10 constructs each co-transfected with mCherry-CRM1. Scale bar: 5 µm. f. FRAP of GFP-NUP98::DDX10 (top), and of mCherry-CRM1 (bottom) in GFP-NUP98::DDX10 condensates in U2OS cells exemplified in b. Data are mean ± s.d. n = 10 in all cases. g. (top) Quantification of GFP intensity in the bleached condensates. (bottom) Control quantification of mCherry intensity in the bleached condensates. Data are mean ± s.d. P-values are from Tukey’s post-hoc test after one-way ANOVA. (Top): P = 0.64; (bottom): P = 0.15. n = 10 cells in all cases from two biologically independent experiments. h. Fixed cell imaging of U2OS cells ectopically expressing GFP-NUP98::DDX10, GFP-NUP98::DDX10-KS and GFP-NUP98::DDX10-KSF-to-G and cleavable mCherry reporter with or without treatment with 5% 1,6-hexanediol. i. Quantification of effect of 1,6-hexanediol to granularity of GFP signal represented by relative standard deviation* of GFP signal in the nuclei (left). Small regions of saturated GFP signal were allowed - see Methods. Mean intensities of cleavable mCherry reporter (middle) and intensities of mean GFP signal outside the detected NUP98::DDX10 foci (right). P-values are from Tukey’s post-hoc test after one-way ANOVA. (Left): P(ND_+HD vs ND_-HD) = < 0.001, P(ND-KS_-HD vs ND_-HD) = 0.35, P(ND-KS_+HD vs ND-KS_-HD) = 0.02, P(ND-KS_F-to-G_-HD vs ND-KS_-HD) = < 0.001, P(ND-KS_F-to-G_+HD vs ND-KS_F-to-G_-HD) = 0.97; (middle): P(ND_+HD vs ND_-HD) = 0.04, P(ND-KS_-HD vs ND_-HD) = < 0.001, P(ND-KS_+HD vs ND-KS_-HD) = 0.86, P(ND-KS_F-to-G_-HD vs ND-KS_-HD) = < 0.001, P(ND-KS_F-to-G_+HD vs ND-KS_F-to-G_-HD) = 0.83; (right): P(ND_+HD vs ND_-HD) = < 0.001, P(ND-KS_-HD vs ND_-HD) = < 0.001, P(ND-KS_+HD vs ND-KS_-HD) = 0.92, P(ND-KS_F-to-G_-HD vs ND-KS_-HD) = < 0.001, P(ND-KS_F-to-G_+HD vs ND-KS_F-to-G_-HD) = 0.23. Data are mean ± s.d. “n” represents the number of cells from two biologically independent experiments.
