Fig. 3: Mutant HMGB1 replaces the granular component of the nucleolus in vivo.
From: Aberrant phase separation and nucleolar dysfunction in rare genetic diseases
a, Representative images of live U2OS cells expressing eGFP–HMGB1 proteins. Nuclear area revealed by Hoechst staining is shown as dashed white lines in a, c and f. b, Model of the nucleolus. R1, RNA polymerase I. c, Left, representative images of U2OS cells expressing RFP–FIB1 and mutant eGFP–HMGB1. Right, fluorescence intensity profiles from the region highlighted by the dashed yellow line. Low and high indicate nuclei with a relatively low or high amount, respectively, of the mutant protein. d, Relative fluorescence intensity of eGFP–HMGB1 before and after photobleaching. Data displayed as the mean ± s.d. e, Schematic and sequence representation of HMGB1 variants. Blue bars, HMG boxes. NLS, nuclear localization signal. Red arrow marks the position of the frameshift mutation (K184Rfs*44) and red letters highlight mutagenized amino acids. f, Representative images of live U2OS cells expressing the indicated eGFP–HMGB1 variants. g, Relative fluorescence intensity of eGFP–HMGB1 variants before and after photobleaching. Data are displayed as a line for the mean signal, with the shaded region representing ± s.d., n = number of cells examined. h, Representative images from puromycin-staining experiments with U2OS cells ectopically expressing eGFP–HMGB1 proteins. The puromycin signal was used to trace the cell area to highlight GFP+ cells with a dashed line. i, Normalized puromycin intensities displayed as the mean ± s.d. from three independent biological replicate experiments. ***P < 0.0002, ****P < 0.0001 by one-way ANOVA. j, Quantification of the viability of cells expressing the indicated HMGB1 proteins. Data displayed as individual points from independent biological replicates (n = 4). Bar charts show mean ± s.d. *** P = 0.0005, * P = 0.0177 by one-way ANOVA. Scale bars, 10 µm (a,c,f) or 20 µm (h).
