Morphological profiling with the Cell Painting assay has emerged as a promising method in drug discovery research. The assay captures morphological changes across various cellular compartments enabling the rapid prediction of compound bioactivity. We present a comprehensive morphological profiling resource using the carefully curated and well-annotated EU-OPENSCREEN Bioactive compounds. The data were generated across four imaging sites with high-throughput confocal microscopes using the Hep G2 as well as the U2 OS cell lines. We employed an extensive assay optimization process to achieve high data quality across the different sites. An analysis of the extracted profiles validates the robustness of the generated data. We used this resource to compare the morphological features of the different cell lines. By correlating the profiles with overall activity, cellular toxicity, several specific mechanisms of action (MOAs), and protein targets, we demonstrate the dataset's potential for facilitating more extensive exploration of MOAs.

Biofarma Research Group Centro de Investigación CIMUS Departamento de Farmacología Facultad de Farmacia Instituto de Investigaciones Sanitarias IDIS Universidade de Santiago de Compostela 15706 Santiago de Compostela Spain

CZ OPENSCREEN National Infrastructure for Chemical Biology Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 14220 Prague 4 Czech Republic

EU OPENSCREEN ERIC Campus Berlin Buch Robert Roessle Str 10 13125 Berlin Germany

Fundación MEDINA Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía Parque Tecnológico Ciencias de la Salud Avda del Conocimiento 34 18016 Armilla Granada Spain

Institute of Molecular and Translational Medicine Palacky University and University Hospital in Olomouc Faculty of Medicine and Dentistry Hněvotínská 5 77515 Olomouc Czech Republic

Leibniz Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e 5 Campus Berlin Buch Robert Roessle Str 10 13125 Berlin Germany

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