Panel A: A series of microscopy images. The images are divided into two columns: Control siRNA and siRNA knockdown. Each row represents a different protein (LRRIQ3, CCAR1, RAD51C, NBN). The images show the staining patterns for the HPA antibody, GM130, and the merged images. The rightmost column contains bar graphs quantifying the intensity measurements in the Golgi and nuclear compartments for each protein. Panel B: A network diagram displaying protein-protein interactions of Golgi-nuclear localization proteins. Proteins are categorized by functional pathways: DNA repair, membrane trafficking, lipid metabolism, RNA metabolism, and cell cycle. Each category is color-coded. Panel C: A scatter plot showing the Pearson's correlation coefficient (PCC) between cis-Golgi and trans-Golgi markers and DDR proteins. The x-axis lists different proteins, and the y-axis shows the PCC values. Two sets of data points are plotted, one for TGN46 and one for GM130.
Validation of DDR protein localization and functional network analysis, related to Fig. 1. (A) Representative images of HeLa-K cells stained with antibodies against DDR proteins and the Golgi marker, GM130. Cells were treated with a control or an siRNA against the protein of interest to validate the specificity of the antibody. The changes in intensity measurements in the Golgi and nuclear compartment were quantified. (n = 2, biologically independent experiments with at least 200 cells analyzed for each treatment.) Scale bar, 10 μm. (B) Experimentally based protein–protein interaction networks displaying the Golgi–nuclear localization proteins validated in this study. Proteins are categorized by functional pathways annotated using gene ontology terms and literature curation (Table S1). (C) Golgi-cisternal localization analysis of Golgi–nuclear DDR proteins. HeLa-K cells were treated with nocodazole (33 μM, 3 h), fixed, and stained with antibodies against DDR proteins. Quantification of PCC between cis-Golgi and trans-Golgi markers and DDR proteins; N ≥ 4 cells; n ≥ 36 mini-stacks.