Fig. 1

High-content image analysis of cell-cycle-dependent peroxisomal signal in U-2OS cells expressing the mOrange2-Peroxisomes2 marker. A Representative image of U-2OS cell line with mOrange2-Peroxisomes2 fluorescent label, scale bar: 100 μm. B High-content image analysis workflow of single-cell detection and peroxisomal signal quantification. Single cell nuclei were detected based on the Hoechst staining (blue markers), while clustered nuclei or the nuclei at the edge of images (yellow markers) were excluded from analysis (primary object identification). Afterward, selected single cells were segmented into cytoplasmic and nuclear regions (nuc/cyto segmentation), and the peroxisomal signal in the cytoplasm was quantified (peroxi spot identification). C The peroxisomal signals after the treatment with compounds (1 μM, 24 h) from the target-selective chemical library were normalized to the control average (DMSO treatment). Blue: compounds reducing peroxisomal signals. Red: compounds increasing peroxisomal signals. D Histogram showing the distribution of Hoechst DNA staining signals in the control cell population. E Peroxisomal spot total intensity was plotted against the Hoechst signal in individual cells of the control sample. The distribution of peroxisomal spot total intensity per cell was summarized in cell populations of different cell cycle phases. F Flow cytometry analysis of U-2OS cells with peroxisomal fluorescent marker and Hoechst staining. The relative levels of peroxisomal intensities in each cell cycle phases were normalized against G1 cell population. G U-2OS cells were treated with aphidicolin (DNA synthesis inhibitor, 1 μM), VX-680 (Aurora Kinase inhibitor, 1 μM) or aphidicolin plus VX-680 for 24 h. The cell cycle distribution and peroxisomal signals were analyzed by flow cytometry. The relative levels of peroxisomal intensities in each sample were normalized against the control cell population. n = 3 independent experiments. *p < 0.05; ***p < 0.001