Extended Data Fig. 3: Characteristics of nucleoli and nucleolar proteins in the presence or absence of Pol II inhibition.
From: Nucleolar RNA polymerase II drives ribosome biogenesis
a, b, Schematic of a nucleolus, illustrating the localization of LLPS nucleolar subcompartments marked by the resident proteins NPM and UBF (a), which are highly disordered, as revealed using the various short long 2 (VSL2) predictor of natural disordered regions (PONDR) algorithm (b). c, Effects of Pol II inhibition (iPol II) on NPM localization, as shown by immunofluorescence microscopy. Examples of normal and defective phenotypes are respectively marked by magenta and white arrowheads. d, Quantification of the percentage of cells that have any NPM phase-separated body reveals that the fast-acting Pol II inhibitor FP completely disrupts nucleoli before the slower-acting Pol II inhibitor AMN can take effect. Not depicted is the percentage of cells with perturbed nucleolar architecture as evidenced by NPM1 ruffling, which increased from 0.6 ± 4.6% to 63.3 ± 5.7% following the 1-hour FP treatment. e, Pol II inhibition also disrupts NPM localization in IMR90 cells. f, Effects of Pol II inhibition on UBF localization, as shown by immunofluorescence microscopy. Examples of normal and defective phenotypes are respectively marked by magenta and white arrowheads. g, Quantification of the percentage of cells that have any punctate UBF localization confirmed that the fast-acting FP completely disrupts nucleoli before the slower-acting AMN. h, Pol II inhibition triggers various aberrant UBF localization phenotypes, as shown in representative images. i, Global nucleolar disruption following Pol II inhibition, as revealed by phase-contrast microscopy. The fraction of cells with more than three black nucleolar bodies is indicated. j, Live-cell UBF fluorescence recovery after photobleaching (FRAP). Mock control cells were continuously imaged without a photobleaching step. FRAP FP/vehicle rate-constant ratio = 2.3. k, Formerly nucleolar space became Congo red positive after Pol II inhibition. c–k, HEK293T cells were used unless otherwise indicated; data are means ± s.d.; one-way ANOVA with Dunnett’s multiple comparisons test, n = 3 biologically independent experiments (d, g) or n = 5 biologically independent experiments (i); for j, vehicle FRAP cells n = 30, vehicle control cells n = 4, FP FRAP cells n = 15, and FP control cells n = 6; images in e, h, k are representative of two independent experiments. Scale bars, 5 μm (yellow) or 1 μm (white).
