Panel A shows a schematic of Giantin (GOLGB1) with coiled coil domains, transmembrane domain, cytoplasmic region, and Golgi lumen. Panel B shows fluorescence images of control small interfering RNA and small interfering Giantin cells stained for RAD51C, GM130, and merged images with nuclei, highlighting RAD51C localization using arrowheads. Panel C shows a bar graph of RAD51C Golgi intensity (arbitrary units) comparing control small interfering RNA, small interfering Giantin, and small interfering GMAP210 treatments. Panel D shows a bar graph of the RAD51C distribution ratio (Golgi per nucleus) comparing control small interfering RNA, small interfering Giantin, and small interfering GMAP210 treatments. Panel E shows a bar graph of RAD51C nuclear foci per cell comparing control small interfering RNA, small interfering Giantin, and small interfering GMAP210 treatments. Panel F shows fluorescence images of Control and Nocodazole treated cells stained for RAD51C, Giantin, and merged images with nuclei. Panel G shows a zoomed fluorescence image and a line graph of Golgi cisternae localization, with the x-axis labeled Distance normalized (micrometer) and the y-axis labeled Fluorescence intensity (percent of maximum), comparing RAD51C and Giantin. Panel H shows immunoprecipitation Western blot analyses using Giantin and RAD51C antibodies with Immunoglobulin G controls. Panel I shows an immunoprecipitation Western blot of Giantin and RAD51C under Doxorubicin, Ataxia telangiectasia mutated inhibitor, and Importazole treatments. Panel J shows fluorescence images of control small interfering RNA and small interfering Giantin cells stained for RAD51C, gamma H2AX, and merged images with nuclei. Panel K shows a bar graph of the percentage of RAD51C co-localization with gamma H2AX comparing control and Etoposide treatments in control small interfering RNA and small interfering Giantin cells. Panel L shows fluorescence images of control small interfering RNA and small interfering Giantin cells stained for RAD51C, phosphorylated Ataxia telangiectasia mutated, and merged images with nuclei. Panel M shows a bar graph of the percentage of RAD51C co-localization with phosphorylated Ataxia telangiectasia mutated comparing control and Etoposide treatments in control small interfering RNA and small interfering Giantin cells.
RAD51C Golgi localization is dependent on the golgin protein, giantin. Immunofluorescence in this figure was performed using RAD51C antibody ab72063; co-IP in H and I used RAD51C antibody ab95069 and giantin antibody AF8159. (A) Schematic diagram highlighting the domain organization of giantin. (B) RAD51C protein redistribution upon depletion of giantin. HeLa-K cells were transfected with either control or giantin siRNAs for 72 h, then immunostained for RAD51C and the Golgi marker GM130. Yellow arrowheads mark the Golgi, and white arrowheads denote RAD51C nuclear foci. Scale bars, 10 μm. (C–E) Quantifications of RAD51C localization following depletion of giantin or GMAP210: (C) total Golgi-associated RAD51C intensity, (D) the Golgi-to-nucleus RAD51C intensity ratio, and (E) the number of nuclear RAD51C foci per cell. Data are presented as mean ± SEM. (n = 3 biologically independent experiments, 1,445 total cells). Statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (F) Golgi-cisternal co-localization analysis. HeLa-K cells treated with nocodazole (33 μM, 3 h), fixed, and stained with antibodies against giantin and RAD51C. Scale bar, 10 μm. (G) Enlarged view of single isolated mini-stack from F; white line across the stack was used for line-scan analysis. Scale bar, 1 μm. (H and I) co-IP of endogenous giantin with RAD51C under control conditions (H) or after DOX treatment and co-treatments with ATM inhibitor (ATMi) and IPZ (I) in HeLa-K extracts. (J–M) Co-localization experiment of cells treated with control siRNA, or giantin siRNA under control conditions or after treatment with ETO. Cells stained with antibodies against RAD51C (green) and HR DDR markers (red): (J) γ-H2AX and (L) p-ATM. Co-localizing foci are indicated by arrowheads. Scale bars, 10 μm. Quantification of percentage RAD51C foci co-localizing with (K) γ-H2AX and (M) p-ATM. Data represent the mean ± SEM. (n = 3 biologically independent experiments with at least 200 cells and at least 550 RAD51C nuclear foci analyzed for the co-localization experiments for each condition). Statistical significance determined by two-tailed Student’s t test. Source data are available for this figure: SourceData F4.