Fig. 3: DVP defines single-cell heterogeneity at the subcellular level.

a, Segmentation of whole cells and nuclei in BIAS of DNA (DAPI)-stained U2OS cells. Scale bar, 20 μm b, Automated LMD of whole cells and nuclei into 384-well plates. Images show wells after collection. c, Relative protein levels (x axis) of major cellular compartments between whole cell (n = 3 biological replicates) and nuclei (n = 3 biological replicates) specific proteomes. y axis displays point density. d, Left: conceptual workflows of the phenotype finder model of BIAS for ML-based classification of cellular phenotypes. Right: results of unsupervised ML-based classification of six distinct U2OS nuclei classes based on morphological features and DNA staining intensity. Colors represent classes. Scale bar, 20 μm. e, Phenotypic features used by ML to define six distinct nuclei classes. Radar plots show z-scored relative levels of morphological features (nuclear area, perimeter, solidity and form factor) and DNA staining intensity (total DAPI signal). f, Example images of nuclei from the six classes identified by ML. Blue color shows DNA staining intensity, and red color shows EdU staining intensity to identify cells undergoing replication. Represented nuclei are enlarged for visualization and do not reflect actual sizes. g, PCA of five interphase classes based on 3,653 protein groups after data filtering. Replicates of classes (n = 3 biological replicates) are highlighted by ellipses with a 95% confidence interval. h, Enrichment analysis of proteins regulated among the five nuclei classes. Significant proteins (515 ANOVA significant, FDR < 0.05, s₀ = 0.1) were compared to the set of unchanged proteins based on Gene Ontology Biological Process (GOBP), Reactome pathways as well as cell cycle and cancer annotations derived from the Human Protein Atlas (HPA)²⁰. A Fisher’s exact test with a Benjamini–Hochberg FDR of 0.05 was used (Supplementary Table 3). i, Unsupervised hierarchical clustering of all 515 ANOVA significant protein groups (Supplementary Table 4). Cell-cycle-regulated proteins reported by the HPA are shown in the lower bar. Nuclei classes (n = 3 biological replicates) are shown in the row bar. C1–C4 show clusters upregulated in the different nucleus classes. j, Network analysis of enriched pathways for protein clusters C1–C4. Pathway enrichment analysis was performed with the ClusterProfiler R package³⁶. ER, endoplasmic reticulum; PC, principal component.