Fig 2: Rad50 promotes migration, invasion and induces EMT of ovarian cancer cells in vitro. (A and B) Migration and invasion were performed in A2780, SKOV3 and HO8910 cells with Rad50 overexpression or knockdown. (C) Morphological changes of A2780 cells with Rad50 overexpression or knockdown. (D) EMT-related markers were detected by western blot in A2780, SKOV3 and HO8910 cells with Rad50 overexpression or knockdown. (E) Immunofluorescence staining showed E-cadherin and N-cadherin expression in SKOV3 cells with Rad50 knockdown. **p<0.01, ***p<0.001, when compared with the control group (Student's t-test)

Fig 3: Rad50 promotes proliferation and anchorage-independent growth in ovarian cancer cells. (A) Effect of Rad50 on clonogenic ability in ovarian cancer cell lines. (B) Soft agar colony formation assay exhibited the effect of Rad50 on anchorage-independent growth and malignant transformation. (C) Growth curves illustrated the cell proliferation in A2780, SKOV3 and HO8910 cells. (D) Cell cycle analysis of synchronized SKOV3 cells with Rad50 knockdown in comparison with control cells measured by flow cytometry after 0, 4- and 8-hours culture in the complete medium. (E) Western blot analysis of cell cycle–related proteins in ovarian cancer cell lines with Rad50 overexpression or knockdown. The data shown here are from a representative experiment repeated three times with similar results. Data are presented as means ±S.D. *p < 0.05, **p<0.01, ***p<0.001, when compared with the control group (Student's t-test)

Fig 4: Rad50-mediated activation of NF-?B pathway is CARD9 dependent. (A) Interaction of Rad50 and CARD9 was determined by co-IP and western blot in HEK293T and ovarian cancer cell lines immunoprecipitated with either anti- Rad50 antibody or IgG control. (B) Interaction of Rad50 and CARD9 was determined by co-IP and western blotting in indicated cell lines immunoprecipitated with anti-CARD9 antibody or IgG control. (C) Transwell and western blot analysis of SKOV3 and A2780 cells transfected with indicated combinations of siRNA or vectors. *p < 0.05, **p<0.01, ***p<0.001, one-way ANONA

Fig 5: Rad50 promotes tumour growth and metastasis of xenografts. (A-B) Photographs illustrated tumours in xenografts via subcutaneous injection of A2780 cells with Rad50 overexpression or knockdown. (C) Representative images of HE staining and IHC staining with indicated antibodies of tumours isolated from mice. (D) Representative images of lungs and HE staining isolated from mice that received tail vein injection. *p < 0.05, **p<0.01, ***p<0.001, student's t-test was performed when comparing 2 groups, one-way ANOVA was performed when comparing multiple groups