Extended Data Fig. 4 |. NORAD-induced PUM phase separation at physiologic concentrations in vitro.

a, Representative 3D-reconstructed confocal image of a PUM1-stained NORAD^−/− HCT116 cell used to estimate cytoplasmic volume. The length (l), width (w), and depth (d) of the entire cell and its nucleus were measured and used to calculate the total cellular and nuclear volumes using the ellipsoid volume formula. Cytoplasmic volume for each cell was determined by subtracting nuclear volume from total volume. b, Box-and-whisker plot of measured cytoplasmic volumes (n = 20 cells). Mean cytoplasmic volume shown on the right. Boxes extend from 25^th to 75^th percentiles; middle line represents median; whiskers extend from minima to maxima. c, Schematic of wild-type and mutant NORAD transcripts. Location of repeated NORAD domains (ND1-ND5) indicated with gray boxes and mammalian sequence conservation shown in green (UCSC Genome Browser hg38 PhastCons track). Location of PREs indicated with red and yellow arrowheads. PREmut transcript contains 18 UGU to ACA mutations in PREs (gray arrowheads). ND4 represents the most conserved segment of NORAD and contains 4 PREs. Figure modified from Elguindy et al.⁵. d, DIC and fluorescence microscopy images of PUM1 (150 nM, green) droplets in the presence or absence of NORAD (2 nM) or PEG₃₃₅₀.