Fig. 2: Schematic overview of PLOM-CON analysis for estimating the AX-53802 MoA.

[Illustrations adapted with modifications from Noguchi et al. (2021), iScience, 24(7)]. A HEK293 cells in 96-well plates treated under three distinct conditions; untreated (control), 0.8 μM AX-53802, and AX-53802 + 1 μM Fer1. Cells were fixed at 5, 10, 15, 20, 25, 30, 40, 50, and 60 min post-treatment. B Immunofluorescence and image analysis were conducted following (A). Antibodies listed in Supplementary Data 1 were used for staining, followed by imaging using a confocal laser-scanning microscope (×40 objective). Segmentation was performed, with Hoechst and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) staining used as nuclear and cell region markers, respectively. Specific masks were created for areas with intense localized staining: “domains” (relatively large), “aggregates” (small), and PM-associated aggregates. Following segmentation, feature quantities were collected, as detailed in Supplementary Data 2, including fluorescence intensity across cellular compartments (nucleus, cytoplasm, domain, aggregates, and PM), domain and aggregate formation rates, and morphological characteristics, e.g., area and circularity. C Covariation network constructed using feature quantities from (B). Partial correlation matrix, viewed as a weighted adjacency matrix, was estimated using the graphical lasso algorithm and visualized as networks (refer to Fig. S5). In the network, nodes represent proteins of interest and subnodes are color-coded by the cellular localizations (nucleus, cytoplasm, domains, and aggregates) of feature quantities. Edges between nodes indicate temporal correlations between protein feature quantities within each localization. D PLOM-CON analysis was conducted under three conditions: negative control (untreated cells), AX-53802 treatment, and AX-53802 + Fer1 cotreatment. Fer1 counteracts lipid peroxide accumulation, offering insights into protein changes preceding or following lipid peroxide formation in the pathway.