Fig. 5: Colocalization study of NPM1 condensates with macromolecules.

a Confocal imaging of mCherry-NPM1 condensates incubated with Alexa Fluor 488-labeled anti-HisTag (positive control) and anti-Biotin (negative control) antibodies at different NPM1 concentrations. Anti-HisTag is selectively recruited into NPM1 condensates, while anti-biotin remains excluded. b Flow cytometry confirms colocalization results from a, showing increased Alexa Fluor 488 fluorescence for anti-HisTag but not for anti-Biotin. Statistical comparisons are made against the 0 µM protein condition. c Confocal imaging of 20 µM NPM1 condensates incubated with 2 µM lipid probes reveals selective partitioning of Oregon Green phosphatidylethanolamine (a lipid-modified dye) but exclusion of Oregon Green 488 (a hydrophilic dye), indicating a preference for lipid-like molecules. d Flow cytometry corroborates the imaging data in c, showing increased Alexa Fluor 488 fluorescence in the presence of Oregon Green phosphatidylethanolamine. Pre-incubation with 2 mg/mL neomycin disrupts lipid association without affecting mCherry-NPM1 condensate formation. e Confocal imaging of 20 μM NPM1 condensates incubated with small-molecule drugs (50 μM mitoxantrone or 100 μM FLTX1) demonstrates their strong partitioning into condensates. f Flow cytometry confirms drug partitioning, showing an increase in fluorescence intensity for both mitoxantrone and FLTX1 in NPM1 condensates. Statistical comparisons are between mitoxantrone versus buffer only (red channel), and FLTX1 versus buffer only (green channel). g Schematic representation of RNA labeling using Alexa Fluor 488. h Confocal imaging shows strong colocalization of RNA-Alexa 488 with NPM1 condensates, indicating RNA recruitment. i Flow cytometry analysis supports confocal imaging in h, demonstrating robust RNA partitioning into NPM1 condensates. Statistical comparisons are between condensates with RNA and condensates without RNA. 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 (***), p < 0.0001 (****) and not significant (ns.) for p ≥ 0.05. Exact p-values are provided in the Source Data file. Scale bars = 5 µm.