Fig. 2: Characterization of condensate formation and dissolution using confocal microscopy and flow cytometry.

a Schematic overview of the in vitro droplet assay workflow. Purified fluorescently tagged proteins (mCherry-NPM1, mCherry-DDX4, mCherry-HP1α, and EGFP-HMGB1) were incubated with 10% PEG8000 to induce condensate formation, followed by treatment with 10% 1,6-hexanediol to assess reversibility. b Confocal images and flow cytometry analysis of 20 µM mCherry-NPM1 under control, PEG, and PEG + hexanediol conditions. PEG treatment induces bright, spherical condensates, which are disrupted by 1,6-hexanediol. Flow cytometry confirms increased fluorescence intensity with PEG and reduction upon hexanediol treatment, indicating reversible condensate formation. c Forward scatter (FSC) versus side scatter (SSC) plots of mCherry-NPM1 with and without PEG. Under equal acquisition time (30 s), PEG treatment resulted in a markedly larger number of detected events (9567 vs. 765). d Histogram of mCherry fluorescence intensity for mCherry-NPM1 with and without PEG. PEG treatment caused a clear rightward population shift, reflecting enhanced condensate-associated signal. e Quantification of mean fluorescence intensity (MFI) of mCherry-NPM1 under no PEG, with PEG, and PEG + 1,6-hexanediol conditions, showing reversible condensate formation and dissolution. f Confocal microscopy images of mCherry-DDX4, mCherry-HP1α, and EGFP-HMGB1 under no PEG, with PEG, and PEG + 1,6-hexanediol conditions. g Flow cytometry MFI analyses of mCherry-DDX4, mCherry-HP1α, and EGFP-HMGB1 under the same conditions. Flow cytometry data represent properly gated populations of fluorescent particles. Statistical analysis was performed using unpaired, two-tailed student’s t-tests. Error bars indicate standard deviation (SD). Significance is denoted as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Scale bars = 5 µm. Exact p-values are provided in the Source Data file.