Cell Painting PLUS imaging data: U2OS biological replicate 4 (imaging cycle 2)

These are supplementary data for Wedler et al. (2026): Cell Painting PLUS: An iterative staining-elution protocol for high-content phenotypic screenings (Current Protocols, doi:10.1002/cpz1.70368 ). The data represent the second imaging cycle of the fourth biological replicate in U2OS cells of the Cell Painting PLUS experiment described in von Coburg et al. 2025 (https://doi.org/10.1038/s41467-025-58765-8). Cells were incubated with compounds for 48 h, stained and imaged with five fields per well and two optical sections (z-planes) using Opera Phenix microscope (Revvity Inc.).Channel Hoechst 33342; (Excitation: 405; Emission: 435-480) depicts DNA (stained with Hoechst 33342).Channel Alexa 488 (Excitation: 488, Emission: 500-550) represents Golgi apparatus (stained with Wheat Germ Agglutinin (WGA)). Channel Alexa 568 (Excitation: 561; Emission: 570-630) shows mitochondria (stained with MitoTracker Orange CMTMROS). Channel Alexa 647 (Excitation: 640; Emission: 650-760) visualizes endoplasmic reticulum (stained with Concanavalin A).The imaging data are presented in the format of a Harmony archive file. The file needs to be imported into the Harmony software (Revvity Inc.) to access the imaging data. The plate layout (including information on compounds and tested concentrations) is described in the Compound_layout.xlsx file. Staining and imaging details are described in in von Coburg et al. 2025 (https://doi.org/10.1038/s41467-025-58765-8).The raw imaging data presented here can be used as input data for further image analysis as described in Wedler et al (2026).