Panel A shows fluorescence images of control small interfering RNA, small interfering Giantin, and small interfering GMAP210 cells treated with Dimethyl sulfoxide or Etoposide, highlighting micronuclei with arrowheads. Panel B shows a bar graph of the percentage of cells with micronuclei after Dimethyl sulfoxide treatment, with the x-axis labeled control small interfering RNA, small interfering Giantin number 1, small interfering Giantin number 2, small interfering GMAP210 number 1, small interfering GMAP210 number 2, small interfering RAD51C and the y-axis labeled percentage of cells with micronuclei. Panel C shows a bar graph of the percentage of cells with micronuclei after Etoposide treatment, with the x-axis labeled control small interfering RNA, small interfering Giantin number 1, small interfering Giantin number 2, small interfering GMAP210 number 1, small interfering GMAP210 number 2 and the y-axis labeled percentage of cells with micronuclei. Panel D shows fluorescence images of control small interfering RNA and small interfering Giantin cells from a comet assay. Panel E shows a bar graph of relative Olive tail moment, with the x-axis labeled control small interfering RNA, small interfering Giantin number 1, small interfering Giantin number 2, small interfering RAD51C and the y-axis labeled relative Olive tail moment. Panel F shows colony formation assay images of HeLa Kyoto cells treated with Dimethyl sulfoxide, comparing control small interfering RNA and small interfering Giantin. Panel G shows a bar graph of colony formation (area percent), with the x-axis labeled control small interfering RNA, small interfering Giantin number 1, small interfering Giantin number 2 and the y-axis labeled colony formation (area percent). Panel H shows colony formation assay images of U-2 OS cells treated with Dimethyl sulfoxide, comparing control small interfering RNA and small interfering Giantin. Panel I shows a bar graph of colony formation (area percent), with the x-axis labeled control small interfering RNA, small interfering Giantin number 1, small interfering Giantin number 2 and the y-axis labeled colony formation (area percent). Panel J shows Western blot analysis of phosphorylated Ataxia telangiectasia mutated and alpha tubulin in control small interfering RNA and small interfering Giantin cells treated with Dimethyl sulfoxide or Doxorubicin. Panel K shows a Western blot analysis of phosphorylated Ataxia telangiectasia mutated and alpha Tubulin in cells treated with Dimethyl sulfoxide, Doxorubicin, and Importazole. Panel L shows a bar graph of percentage relative homologous recombination rate, with the x-axis labeled negative control, control small interfering RNA, small interfering Giantin, small interfering RAD51C, small interfering Giantin plus small interfering RAD51C and the y-axis labeled percentage relative homologous recombination rate. Panel M shows Western blot analyses of phosphorylated Ataxia telangiectasia mutated, Giantin, RAD51C, and alpha tubulin in cells treated with Dimethyl sulfoxide, Doxorubicin, or Etoposide under different small interfering RNA conditions. Panel N shows a bar graph of relative levels of phosphorylated Ataxia telangiectasia mutated, with the x-axis labeled Doxorubicin and Etoposide treatment groups and the y-axis labeled relative levels of phosphorylated Ataxia telangiectasia mutated. Panel O shows a bar graph of micronuclei percentage incidence, with the x-axis labeled control small interfering RNA, small interfering Giantin, small interfering RAD51C, small interfering Giantin plus small interfering RAD51C and the y-axis labeled micronuclei percentage incidence. Panel P shows a bar graph of fold change in cell number, with the x-axis labeled control small interfering RNA, small interfering Giantin, small interfering RAD51C, small interfering Giantin plus small interfering RAD51C and the y-axis labeled fold change in cell number. Panel Q shows a schematic model illustrating the spatiotemporal regulation of homologous recombination by RAD51C and Giantin, depicting double stranded DNA break, DNA damage response activation and regulation, DNA repair, Golgi, phosphorylated Ataxia telangiectasia mutated, Importin beta dependent transport, and RAD51C interactions.
Depletion of giantin impairs ATM signaling and HR efficiency, increasing genomic instability and cell proliferation. (A) Representative images of HeLa-K cells with nuclei labeled with Hoechst 33342 following siRNA treatment. Yellow arrowheads denote micronuclei structures. Scale bar, 10 μm. (B and C) Quantification of micronuclei formation following siRNA treatment under (B) basal conditions and (C) after ETO-induced DNA damage (10 μM; 16 h followed by 6-h recovery). Note: siRAD51C is not shown in panel C as RAD51C depletion combined with ETO treatment resulted in insufficient cell viability for reliable quantification. Data represent the mean ± SEM (n = 3 biologically independent experiments with >10,000 cells analyzed). Statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (D) Representative comet assay images measuring genomic DNA fragmentation in HeLa-K cells treated with control, giantin, or RAD51C targeting siRNA. Scale bar, 50 μm. (E) Quantification of Olive tail moment from comet assay. Data represent the mean ± SEM (n = 3 biologically independent experiments). Statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (F–I) Colony formation assays in HeLa-K (F and G) and U-2 OS (H and I) cells transfected with control or giantin siRNAs. Representative images (F and H) and quantification of colony area coverage (G and I). Data represent the mean ± SEM (n = 3 biologically independent experiments). Statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (J) Representative western blot analysis of HeLa-K cells transfected with control and giantin siRNA and treated with DOX (40 μM; 3 h). Extracts were prepared and immunoblotted as indicated (n = 2 biologically independent experiments). (K) Western blot of HeLa-K cells treated with DOX (40 μM; 3 h) and IPZ (20 μM; 3 h). (n = 2 biologically independent experiments). (L) DR-GFP reporter assay measuring HR efficiency following siRNA-mediated depletion of giantin, RAD51C, or both. HEK293T-DR-GFP cells were transfected with the indicated siRNAs for 72 h, followed by transfection of the I-SceI expression plasmid pCBASce for 48 h. Cells transfected with siRNAs but without pCBASce served as a no-DSB background control (shown as negative control). GFP-positive cells were quantified by automated high-content imaging, and the percentage of GFP-positive cells was normalized to the nontargeting siRNA control set to 1. Data represent the mean ± SEM (n = 3 biologically independent experiments). Statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (M and N) Western blot of HeLa-K cells transfected with siRNAs against giantin, RAD51C, or a combination of both followed by treatment with DMSO, DOX (40 μM; 3 h) (M), or ETO (10 μM; 3 h) (N) with associated quantification showing the relative levels of pATM; (n = 3 biologically independent experiments); data represent the mean ± SEM statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (O and P) Quantification of micronuclei formation and fold change in cell number after siRNA treatments (mean ± SEM, n = 3, ≥1,000 cells per condition). Statistical significance determined by one-way ANOVA with Tukey’s post hoc test. (Q) Proposed model for the regulation of HR-mediated repair through the activation of RAD51C at the Golgi complex. RAD51C, a regulatory HR protein, is anchored to the Golgi through its interaction with the cytoplasmic tail of giantin, in response to DSBs; this RAD51C Golgi population redistributes to form nuclear foci. This response requires importin-β–mediated nuclear import and the phosphorylation of ATM protein kinase. We propose that the Golgi functions as a spatiotemporal timing module that gates RAD51C (and other DDR factors) availability to ensure proper HR and DNA-damage signaling. Source data are available for this figure: SourceData F5.