Extended Data Fig. 1: Developing compressed screening by testing 316 small molecules in the U2OS cell line with a Cell Painting readout.

a. Histogram of the log₁₀ Mahalanobis Distance (MD) values calculated for each small molecule perturbation relative to the mean of the negative controls (DMSO) over morphological features quantified by the Cell Paiting assay at 6 hours, 24 hours, and 28 hours. For each time point, the coefficient of variation (std./mean) of the log₁₀MD is reported. b. Histogram of log₁₀MD values calculated for each small molecule perturbation relative to the mean of the negative controls (DMSO) over morphological features quantified by the Cell Paiting assay at the 24-hour timepoint at three drug doses: 0.1, 1, and 10 µM. For each dose, the coefficient of variation (std./mean) of the log₁₀MD is reported. c. Histogram of cell number distribution across 192 DMSO control wells for all imaging plates. d. Histogram of cell number distribution in control (right) and perturbation (left) wells, stratified by drug dosage in the GT. e. Violin plot of cell number for perturbations associated each ground truth (GT) phenotypic cluster, plotted by GT phenotypic cluster. f. Scatterplot of non-zero enrichment scores from one-sided permutation test for each perturbation in each GT phenotype. g. Representative Cell Painting images from the morphological phenotype clusters identified in the GT screen. Cells were stained with dyes marking nuclei (Hoechst 33342), endoplasmic reticula (Concanavalin A-AlexaFluor 488), mitochondria (MitoTracker Deep Red), F-actin (Phalloidin-AlexaFluor 568), Golgi apparati and plasma membranes (Wheat Germ Agglutinin-AlexaFluor 594), and nucleoli/cytoplasmic RNA (SYTO14). Diverse phenotypes were observed in compounded-treated U2OS cells, such as: altered nuclei shape and size (Cluster 7); redistribution of ER to one side of nucleus (Cluster 6); mitochondrial puncta (Clyster 8); and, bright, abundant Golgi staining (Cluster 2). Scale bars: 200 μm. h. Bar plot of fingerprint drug categories and their representation in each cluster. i. Representative images of drugs (anti-neoplastic agents) associated with phenotypic cluster 7 with a range of effect sizes (MDs) in the GT screen. The extent of variation in staining of DNA (Hoechst 33342) and nucleolus and cellular RNA (SYTO14) correlate with MD. Scale bars: 200 μm.