Extended Data Fig. 5: Mutant HMGB1 forms nuclear inclusions in human cells.
From: Aberrant phase separation and nucleolar dysfunction in rare genetic diseases
(a) Quantification of the nuclear enrichment of EGFP as the ratio of mean signal intensities inside and outside the nucleus. Red dashed line depicts a value of 1 (no enrichment). For all boxplots in this figure, the median is shown as a line within the boxplot, which spans from 25th to 75th percentiles. Whiskers depict a 1.5x interquartile range. **** P < 2.2 x 10−16, two-tailed Welch’s t-test. (b) Quantification of nuclear inclusions as the standard deviation of nuclear EGFP fluorescence intensity normalized by mean intensity. **** P < 2.2 x 10−16, two-tailed Welch’s t-test. (c) (left) Graph plotting the intrinsic disorder of HMGB1 predicted by PONDR VLXT algorithm. The positions two different IDR definitions are highlighted with orange bars and the position of HMG boxes with blue bars. IDR1 begins from Asn135 as defined by PONDR analysis and IDR2 begins from Ala164, excluding any sequence belonging to HMG box. (middle) Representative image from U2OS cells expressing EGFP-HMGB1-mutant-IDR2. Scale bar = 10 µm. (right) Relative fluorescence intensity of bleached EGFP-HMGB1 WT full-length, mutant full length and mutant IDRs 1 and 2 before and after photobleaching. Data is displayed with a line showing the mean and lighter shade represents ± SD. (d) Representative images of live MCF7, HCT116 and HEK293T cells expressing mEGFP-HMGB1 variants. The nuclear area is shown as dashed white lines. Scale bar = 10 µm. (e) Representative images of EGFP-HMGB1 within live U2OS cell nuclei before and after photobleaching. FRAP recovery quantified in (Fig. 3d). Scale bar = 1 µm. (f) Representative images of formaldehyde-fixed U2OS cells ectopically expressing EGFP-HMGB1 WT or mutant proteins. Scale bar = 5 µm. (g) Quantification of presence of nuclear inclusions in fixed cells in panel (f) represented as the standard deviation of nuclear EGFP fluorescence intensity normalized by mean intensity. (h) Immunofluorescence for RNAPII, MED1, SC35, HP1α, NPM1 and FIB1 in U2OS cells expressing full length mutant EGFP-HMGB1. (low) indicates a nucleus with a relatively low amount of the mutant protein, (high) indicates a nucleus with a relatively high amount of the mutant protein. Scale bar = 10 µm. (i) Quantification of the ratio between intra- /extranucleolar NPM1 intensity and EGFP-HMGB1 intensity inside nucleoli. r = Pearson’s correlation coefficient, P-value from a two-tailed t-test. (j) Quantification of the average NPM1 fluorescence outside the nucleoli and EGFP-HMGB1 intensity inside the nucleoli for the IF experiments shown in panel (h). r = Pearson’s correlation coefficient, p-value from a two-tailed t-test. (k) Quantification of nuclear inclusions in the panel of EGFP-HMGB1 mutants (Fig. 3e–g) as the standard deviation (SD) of nuclear EGFP signal normalized to the mean nuclear EGFP signal intensity. FL = full length.
