Extended Data Fig. 6: Additional characterization of HMGB1 mutant nuclear inclusions in U2OS cells.

(a) qRT-PCR analysis of rRNA species in U2OS cells expressing wild type and mutant HMGB1 variants. rRNA levels are normalized against an RNAPII transcript (actin), and the values are normalized against the rRNA/actin level measured in the cells expressing wild type HMGB1. Data is shown as mean +/− SD, * p < 0.05, two-tailed Student’s t-test. (b) Representative images from Puromycylation experiments with U2OS cells ectopically expressing EGFP-tagged WT or mutant HMGB1 proteins with (Puro +) and without (Puro -) Puromycin pulse labeling. Scale bar = 20 µm. (c) Histograms depicting % of cells and their normalized puromycin intensities from EGFP+ and EGFP- cells ectopically expressing WT or Mutant full length HMGB1 combined from three independent puromycylation experiments. (d) (top) Scheme of the viability experiment. (bottom) Representative mages of U2OS cells at the end of viability experiments (Fig. 3j). Scale bar = 50 µm. (e) Representative live cell imaging of U2OS cells with doxycycline inducible overexpression of EGFP-HMGB1 variants. Dashed lines show nuclear area defined by Hoechst staining. Scale bar = 10 µm. (f) Quantification of nuclear inclusions as the standard deviation (SD) of nuclear EGFP signal normalized to the mean nuclear EGFP signal intensity. **** P < 2.2 x 10⁻¹⁶, two-tailed Welch’s t-test, n = number of nuclei examined for each condition. (g) Relative fluorescence intensity of EGFP-HMGB1 before and after photobleaching with identical laser settings in cells described in (panels e-f). Data displayed as mean ± SD. (h) Representative images of EGFP-HMGB1 within live U2OS cell nuclei before and after photobleaching with identical laser settings, FRAP recovery quantified in (panel g). Scale bar = 2 µm. (i) (top) Scheme for experiments testing the viability of U2OS cells with Doxycycline-inducible expression of HMGB1 variants. (bottom) representative images from viability experiments 48h after sorting for GFP+ cells. Scale bar = 100 µm. (j) Quantification of viability of cells expressing the indicated HMGB1 proteins. Mean relative light units (RLU) displayed as individual points from independent biological replicate experiments (n = 5 for WT and Mutant, 4 for “Patchless”). Bar charts show the mean ± SD. p-values are from one-way ANOVA.