Image-based single-cell phenotype profiling and analysis of the effects of microtubule inhibitors on mitotic cell profiles. (A) Schematic of the single-cell phenotype profiling pipeline. (B and C) Representative methods for characterizing Golgi morphology. Golgi morphology was quantified by measuring fluorescence peaks (puncta) in GM130-stained images. (B) Peak distribution and (C) the nucleus–Golgi distance were assessed. Scale bars, 20 µm. (D) Characterization of mitotic spindle structure via fluorescence peak extraction. Fluorescence peaks from α-tubulin images were used to distinguish bipolar, multipolar, or monopolar spindle structures. (E) UMAP plot of cell phenotype profiles. Three experimental conditions are shown in color-coded form (top left) or individually highlighted for clarity. Each point represents a single-cell profile. (F and G) DBSCAN clustering results. The two identified clusters are color-coded in F, and these colors are overlaid on an original image of DMSO-treated cells (G). Based on this mapping and subsequent analysis, clusters 0 and 1 correspond approximately to interphase and mitotic cells, respectively. Scale bar, 50 µm. (H) Projection of key features distinguishing interphase and mitotic cells onto the UMAP plot. Here, “mask_area” and “circularity” represent the area and circularity of the cell mask. (I) Features reflecting the effects of drug treatment on mitotic cells. Violin plots (with dashed lines indicating the median and quartiles) display aspect_nuc (aspect ratio of ellipse-fitted chromosomes), mean_nuc (mean DAPI fluorescence intensity), and peaks_num_atubulin (number of fluorescence peaks from α-tubulin staining). (J) Features showing the impact of drug treatment on interphase cells. Measured parameters include area_nuc (nuclear size), objects_num_golgi and len_mean_golgi (number and mean length of skeletonized Golgi structures), and mean_Golgi (mean fluorescence intensity of GM130 staining).