Fig. 1: Characterization of cell injury compounds using cell painting.

Compounds associated with cell injury were each profiled by cell painting (CP) after 24 h of compound exposure in U-2 OS cells. a Active CP compounds (by Mahalanobis distance) are enriched for decreased cell number in the Molecular Libraries Initiative (MLI) dataset²⁵. Red highlight: compounds with the highest CP activity generally have the lowest relative cell numbers. b Cell injury compounds cause distinct CP phenotypes. Left: PCA plots showing unsupervised hierarchical clustering of CP phenotypes into nine clusters (some annotated when grossly associated with a compound category). Compounds causing gross cellular injury that could not be grouped into a more distinct MoA occupied with the largest area in the PCA plot. Right: reduced feature summaries for each cluster (all compound concentrations); rad dist, radial distribution. c “Dot plot” summary of cell injury compound categories by each CP cluster. The dot locations denote cluster identity, the dot sizes denote abundance within each compound subset, and the colors denote average activity score for all compounds in the subset. d The CP clusters of cell injury compounds correlate with cell number. Cluster 9, which is most associated with gross cell injury, has the highest activity score and lowest relative cell number. The cut-off is 3 SD from the mean of DMSO-treated wells using the CP activity (Mahalanobis distances). e Compounds from the MLI dataset with high correlation (MLI-HC) to gross injury signature (cluster 9) are active upon re-testing (red), whereas compounds with no correlation to cluster 9 were not active upon re-testing (blue). Inset: heatmap and dendrogram shows pairwise correlation coefficients between each MLI CP compound profile and each of the nine clusters (red arrowhead, enrichment of cluster 9). f Select CP profiles of cellular injury compounds, demonstrating several different CP phenotypes of cell injury compounds. Rainbow plots denote assigned cluster at each compound concentration; arrow indicates compound concentration of representative image. For rainbow plots, note that phenotypic trajectories do not have to progress through each cluster before reaching the cluster 9 “gross injury” phenotype (dotted lines). Image scales: 50 μm. Data are mean ± SD of four intra-run technical replicates each performed on separate microplates. Source data are provided as a Source Data file.