Panel A shows microscopy images of cells stained for TOPORS, GM130, and TGN46. The images display the localization of TOPORS in relation to the cis-Golgi marker GM130 and the trans-Golgi marker TGN46. Panel B is a line graph showing the fluorescence intensity profiles across individual mini-stacks, with the x-axis representing the normalized distance and the y-axis representing the fluorescence intensity as a percentage of the maximum. The graph includes lines for TOPORS, GM130, and TGN46. Panel C shows images of cells stained for CCAR1, GM130, and TGN46, similar to Panel A. Panel D is a line graph similar to Panel B, showing the fluorescence intensity profiles for CCAR1, GM130, and TGN46. Panel E is a heatmap displaying the Pearson's correlation coefficients between the cis-Golgi and trans-Golgi markers and various DDR proteins. The heatmap includes rows for different DDR proteins and columns for cis and trans Golgi markers, with color indicating the correlation strength. Panel F shows representative images of cells treated with DMSO, DOX, or H2O2, stained for DDR proteins (LIG1, POLQ, CCAR1) and the Golgi marker GM130. The images highlight the localization changes of DDR proteins upon DNA damage. Panel G is a dot plot quantifying the Golgi-nucleus distribution ratio of DDR proteins after treatment with DOX. The x-axis lists the DDR proteins, and the y-axis shows the log10(Golgi/Nucleus ratio) normalized to control. The box plot includes categories for trans-Golgi, cis-Golgi, and no marked correlation. Panel H is a dot plot similar to Panel G, quantifying the Golgi-nucleus distribution ratio of DDR proteins after treatment with H2O2.
Systematic analysis of Golgi–nuclear DDR proteins localization. (A–D) 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 the cis-Golgi marker GM130, the trans-Golgi marker TGN46, and DDR proteins. An enlarged view of a single isolated mini-stack stained for TOPORS (A) and CCAR1 (C) are shown; white lines across the stack indicate the regions used for line-plot analyses shown in B and D. Scale bars, 1 μm. (E) Quantification of PCC between cis-Golgi and trans-Golgi markers and DDR proteins; n ≥ 36 mini-stacks. (F) Representative images of localization changes in dual-localizing DDR proteins upon DNA damage. HeLa-K cells were treated with DOX (40 μM, 3 h) or H2O2 (50 μM for 20 min, followed with 15-min recovery), fixed, and stained with antibodies against DDR proteins (LIG1, POLQ, and CCAR1) and the Golgi marker, GM130. Yellow arrowheads denote the Golgi membranes. Scale bars, 10 μm. (G and H) Quantification of DDR protein Golgi–nuclear distribution ratio after treatment with (G) DOX or (H) H2O2. Data represent the mean ± standard error of the mean (SEM) (n = 3 biologically independent experiments with at least 200 cells analyzed for each protein in control and treatment conditions). Proteins are classified according to their Golgi localization patterns from E: orange, co-localizing with the trans-Golgi marker TGN46; blue co-localizing with the cis-Golgi marker GM130; grey, no marked correlation with either markers. Statistical significance was determined using a two-tailed unpaired Student’s t test; ns, **P < 0.01, ***P < 0.001, and ****P < 0.0001, compared with untreated control.