Figure 4.

BORA ablation impairs growth of tumor cells in vivo and viability of patient- derived spheroids ex vivo. (A) Tumor growth of SK-OV-3 pTRIPZ_BORAV1 xenotransplant transduced cells untreated (grey line) or after a sustained treatment with doxycycline supplemented in drinking water (red line) (n = 7 mice/group). Two-way analysis of variance (ANOVA) was used to calculate the significance (p-value) of the difference between the untreated and doxycycline treated group. * p < 0.05; **** p <0.0001. (B) Representative tumors collected at the time of euthanasia. Bar: 1 cm. (C) Average weight of the tumors. (D) Immunoblot analysis of BORA and tRFP protein levels in 4 representative xenograft tumors from both experimental groups. β-Actin was used as loading control. (E) Immunohistochemical analysis of H&E and Ki67 staining. Representative microscopic stained-images of the OC xenografts from the doxycycline-untreated and treated groups. Ki67 percentage as mean and standard deviation are included in the images for both groups (F) Representative images of the three OC patient-derived ascitic cells grown under anchorage independent conditions with shCTL and shBORA lentiviral transduction. Scale bar: 100 μm. (G) The number of spheres was scored 36 h post-transduction with shCTL and shBORA in the indicated ascitic primary cells, classified according to the diameter (50–100 μm, ≥100 μm and ≥200 μm). (H) Viability assay (MTS) was performed at 96 h post-transduction with shCTL or shBORA in the indicated OC patient-derived ascitic cells. (I) Spheres were used for protein extraction and immunoblot analysis with the indicated apoptosis antibodies 96 h post-transduction. Graphs represent an average of three independent experiments ± SEM. β-Actin was used as loading control. For figure C, E, G and H P-values were calculated using a two-tailed Student’s t-test. * p < 0.05; ** p < 0.01; **** p < 0.0001.