Panel A shows fluorescence images of HeLa Kyoto, U-2 OS, and MCF7 cells stained for RAD51C, GM130, and merged images with zoomed regions, highlighting RAD51C, GM130, and nuclei. Panel B shows a bar graph of RAD51C cellular distribution (percent) across Golgi, nucleus, and cytoplasm in HeLa Kyoto, U-2 OS, and MCF7 cells. Panel C shows a Western blot of RAD51C, GM130, Lamin B1, and alpha-tubulin in Membrane, Nuclear, and Cytoplasmic fractions, with molecular weights in kilodaltons. Panel D shows a bar graph of RAD51C cellular distribution (percent) across membrane, nuclear, and cytoplasmic fractions. Panel E shows fluorescence images of cells treated with Dimethyl sulfoxide or Doxorubicin for 1, 2, 3, and 4 hours, stained for RAD51C, GM130, nuclei, merged images, and zoomed regions. Panel F shows a bar graph of RAD51C Golgi intensity (arbitrary units) following Dimethyl sulfoxide or Doxorubicin treatment over time. Panel G shows a bar graph of RAD51C nuclear intensity (arbitrary units) following Dimethyl sulfoxide or Doxorubicin treatment over time. Panel H shows a bar graph of RAD51C cytoplasmic intensity (arbitrary units) following Dimethyl sulfoxide or Doxorubicin treatment over time. Panel I shows a bar graph of the RAD51C distribution ratio (Golgi to nucleus) following Dimethyl sulfoxide or Doxorubicin treatment over time. Panel J shows a bar graph of the RAD51C distribution ratio (Golgi to nucleus) comparing Dimethyl sulfoxide and Doxorubicin. Panel K shows fluorescence images of cells treated with Dimethyl sulfoxide, Doxorubicin, or Doxorubicin plus Importazole, stained for RAD51C and GM130, with enlarged inset regions. Panel L shows a bar graph of the RAD51C distribution ratio (Golgi to nucleus) comparing Dimethyl sulfoxide, Doxorubicin, and Doxorubicin plus Importazole. Panel M shows a bar graph of RAD51C Golgi intensity (arbitrary units) comparing Dimethyl sulfoxide, Doxorubicin, and Doxorubicin plus Importazole. Panel N shows fluorescence images of cells treated with Dimethyl sulfoxide, Doxorubicin, or Doxorubicin plus Ataxia telangiectasia mutated inhibitor, stained for RAD51C and GM130, with enlarged inset regions. Panel O shows a bar graph of the RAD51C distribution ratio (Golgi to nucleus) comparing Dimethyl sulfoxide, DNA-dependent protein kinase inhibitor, Ataxia telangiectasia mutated inhibitor, and Ataxia telangiectasia and Rad3-related inhibitor under Dimethyl sulfoxide and Doxorubicin treatments. Panel P shows a bar graph of RAD51C Golgi intensity (arbitrary units) comparing Dimethyl sulfoxide, DNA-dependent protein kinase inhibitor, Ataxia telangiectasia mutated inhibitor, and Ataxia telangiectasia and Rad3-related inhibitor under Dimethyl sulfoxide and Doxorubicin treatments.
Redistribution of RAD51C Golgi fraction is required for the formation of RAD51C nuclear foci and is dependent on the kinase ATM. Immunofluorescence in this figure was performed using RAD51C antibody ab72063; biochemical fractionation in C used RAD51C antibody ab95069. Antibody specificity is shown in Fig. S6. (A) Representative images of HeLa-K, U-2 OS, and MCF7 cells stained with antibodies against RAD51C (green) and GM130 (red). DNA stained with Hoechst 33342 (blue). Scale bars, 10 μm (overview), 5 μm (zoomed images). (B) Quantification of RAD51C distribution across the Golgi, nucleus, and cytoplasm in the cell lines shown in A measured from immunofluorescence images. (C) Western blot analysis showing the subcellular membrane (M), nuclear (N), and cytoplasmic (C) fractions of RAD51C. Compartment markers: GM130 (Golgi membranes), Lamin B1 (nuclear), and α-tubulin for the (cytoplasmic). (D) Quantification of western blot analysis (C), showing the distribution of RAD51C across 3 cell fractions: membrane, nuclear, and cytoplasmic; n = 3 biologically independent experiments; data represent the mean ± SEM. (E) Representative images of HeLa-K cells treated with DOX (40 μM), for increasing lengths of time. Yellow arrowheads denote the Golgi membrane; white arrowheads denote nuclear foci. Scale bars, 10 μm (overview), 10 μm (zoomed images). (F–I) Quantification of RAD51C sum intensity at the Golgi (F), in the nucleus (G), and in the cytoplasm (H), and RAD51C Golgi–nuclear distribution ratio (I), following DOX treatment. Data represent the mean ± SEM (n = 3 biologically independent experiments with a total of 2,343 cells analyzed). Statistical significance determined by one-way ANOVA with Dunnett’s post hoc test vs. DMSO control. (J) Quantification of RAD51C membrane-nuclear distribution ratio after DOX treatment, calculated from subcellular fractions (Fig. S6 D). (K) Representative images of HeLa-K cells treated with DMSO or IPZ (20 μM) prior to a 3-h DOX treatment. Yellow arrowheads denote the Golgi membranes; white denote nuclear foci. Scale bars, 10 μm (overview), 5 μm (zoomed images). (L and M) Quantification of RAD51C Golgi–nuclear distribution ratio (L) and relative RAD51C Golgi intensity (M) following IPZ and DOX treatment. Data represent the mean ± SEM. (n = 3 biologically independent experiments; 445 total cells analyzed). Statistical significance determined by two-tailed unpaired Student’s t test. (N) Representative images of HeLa-K cells treated with DMSO, ATM phosphorylation inhibitor (KU55933; 30 μM), ATR inhibitor (VE-821; 10 μM), or DNA-PK inhibitor (NU7441; 10 μM) prior to a 3-h DOX treatment. Yellow arrowheads denote the Golgi membrane. Scale bars, 10 μm (overview), 5 μm (zoomed images). (O and P) Quantification of RAD51C Golgi–nuclear distribution ratio (O) and relative RAD51C Golgi intensity (P) following kinase inhibitor and DOX treatment. Data represent the mean ± SEM (n = 3 biologically independent experiments; 1,679 total cells analyzed). Statistical significance determined by two-tailed unpaired Student’s t test comparing DMSO and DOX conditions within each inhibitor treatment. Source data are available for this figure: SourceData F3.