Panel A shows fluorescence images of cells treated with Dimethyl sulfoxide or Camptothecin, stained for RAD51C and GM130, with yellow arrowheads indicating Golgi-localized RAD51C. Panel B shows a bar graph of RAD51C sum intensity (arbitrary units) in the Golgi and Nucleus following Dimethyl sulfoxide or Camptothecin treatment. Panel C shows a bar graph of the RAD51C distribution ratio comparing Dimethyl sulfoxide and Camptothecin treatments. Panel D shows fluorescence images of cells treated with Dimethyl sulfoxide or Etoposide, stained for RAD51C and GM130, with yellow arrowheads indicating Golgi-localized RAD51C. Panel E shows a bar graph of RAD51C sum intensity (arbitrary units) in the Golgi and Nucleus following Dimethyl sulfoxide or Etoposide treatment. Panel F shows a bar graph of the RAD51C distribution ratio comparing Dimethyl sulfoxide and Etoposide treatments. Panel G shows fluorescence images of cells treated with Dimethyl sulfoxide or Mitomycin C, stained for RAD51C and GM130, with yellow arrowheads indicating Golgi-localized RAD51C. Panel H shows bar graphs of RAD51C sum intensity (arbitrary units) in the Golgi and Nucleus, and the RAD51C distribution ratio, comparing Dimethyl sulfoxide and Mitomycin C treatments. Panel J shows a horizontal bar graph of relative homologous recombination following knockdown of the indicated small interfering RNAs, with a dashed reference line at 1.0. Panel K shows a horizontal bar graph of the normalized percent population with phosphorylation of H2AX following knockdown of the indicated small interfering RNAs, with a dashed reference line at 1.0. Panel L shows fluorescence images of cells treated with control small interfering RNA, small interfering Giantin 1, or small interfering Giantin 2, stained for Giantin and nuclei. Panel M shows a bar graph of cell population (percent) in G1, S, and G2/M phases comparing control small interfering RNA and small interfering Giantin. Panel N shows a Kaplan–Meier survival plot comparing low and high GOLGB1 (201056_at) expression groups, with the x-axis labeled Time (months) and the y-axis labeled Probability. Panel O shows a Kaplan–Meier survival plot comparing low and high GOLGB1 (Q14789) expression groups, with the x-axis labeled Time (months) and the y-axis labeled Probability. Panel P shows a Kaplan–Meier overall survival plot comparing Low Golgb1 Group and High Golgb1 Group, with the x-axis labeled Months and the y-axis labeled percent survival. An inset reports the hazard ratio, log-rank P value, and the number of patients in each group.
RAD51C protein localization and redistribution following treatment with additional DNA damage agents, related to Fig. 3, impact of giantin and other golgins on DNA repair, cell cycle, and patient survival, related to Figs. 4 and 5. (A) Representative images of HeLa-K cells stained with antibodies against RAD51C and GM130 after treatment with CPT (0.1 μM) for 16 h followed by media change for 2 h. Yellow arrowheads denote the Golgi membrane. Scale bar, 10 μm. (B) Quantification of RAD51C distribution between the Golgi and nuclear compartments after CPT treatment. (C) Quantification of RAD51C distribution ratio between the Golgi and nuclear compartments after CPT treatment. (D) Representative images of HeLa-K cells stained with antibodies against RAD51C and GM130 after treatment with ETO (50 μM) for 16 h followed by media change for 2 h. Yellow arrowheads denote the Golgi membrane. Scale bar, 10 μm. (E) Quantification of RAD51C distribution between the Golgi and nuclear compartments after ETO treatment. (F) Quantification of RAD51C distribution ratio between the Golgi and nuclear compartments after ETO treatment. (G) Representative images of HeLa-K cells stained with antibodies against RAD51C and GM130 after treatment with MMC (5 μM) for 16 h followed by media change for 2 h. Yellow arrowheads denote the Golgi membrane. Scale bar, 10 μm. (H) Quantification of RAD51C distribution between the Golgi and nuclear compartments after MMC treatment. (I) Quantification of RAD51C distribution ratio between the Golgi and nuclear compartments after MMC treatment. Data represent the mean ± SEM (n = 3 biologically independent experiments with more than 600 cells analyzed per treatment). Statistical significance was determined using a two-tailed unpaired Student’s t test comparing treated with untreated control. **P < 0.01; ***P < 0.001. (J) Reanalyzed siRNA screen data (Adamson et al., 2012) showing the relative HR repair rate upon systematic knockdown of the golgin protein family (grey) and HR complex proteins (black). The dataset is normalized to the negative control set at 1. (K) Reanalyzed siRNA screen data (Paulsen et al., 2009) showing the relative percent cell population with phosphorylation of H2AX upon systematic knockdown of the golgins. The datasets are normalized to the negative control set at 1. (L) HeLa-K cells were transfected with control, or giantin siRNAs for 72 h. The cells were stained with antibodies against giantin; nuclei were stained with Hoechst 33342. Scale bar, 10 μm. (M) Cell cycle profile of HeLa-K cells treated with giantin or control siRNA. Data represent the mean ± SEM. (n = 3 biologically independent experiments). (N and O) Kaplan–Meier survival plots in patients with high and low giantin expression in breast cancer, (N) at mRNA level (Győrffy, 2021) and (O) protein level (Ősz et al., 2021). Plots were generated using the KM Plotter database (https://kmplot.com). (P) Pan-cancer overall survival map for GOLGB1 expression across all available TCGA cohorts, generated using GEPIA2 (Tang et al., 2019). Tumor type–specific hazard ratios, sample sizes, and P values are provided in Table S3.