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 first imaging cycle of the second 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 actin structures ( stained with Alexa Fluor™ Plus 405 Phalloidin dye).Channel Alexa 488 (Excitation: 488, Emission: 500-550) represents RNA (stained with SYTO™ 14 Green Fluorescent Nucleic Acid Stain). Channel Alexa 568 (Excitation: 561; Emission: 570-630) shows mitochondria (stained with MitoTracker Orange CMTMROS). Channel Alexa 647 (Excitation: 640; Emission: 650-760) visualizes lysosomes (stained with Cell Navigator™ Lysosome Staining Kit).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).