Fig 2: TPX2 knockdown suppresses the proliferation of breast cancer cells by downregulating NCOA5. (A) MCF7 cells were transfected with Ov-NCOA5 or Ov-NC. The transfection efficiency of si-NCOA5 in MCF7 cells was validated via RT-qPCR. ***P<0.001. (B) MCF7 cells were transfected with si-TPX2 or co-transfected with si-TPX2 and Ov-NCOA5. Cell Counting Kit-8 assay for determination of the viability of MCF7 cells. ***P<0.001 vs. si-TPX2. ##P<0.01, ###P<0.001 vs. si-TPX2 + Ov-NC. (C) MCF7 cells were transfected with si-TPX2 or co-transfected with si-TPX2 and Ov-NCOA5. Western blot analysis for determination of Ki67 and PCNA protein expression in MCF7 cells. **P<0.01, ***P<0.001. NC, negative control; NCOA5, nuclear receptor coactivator 5; OD, optical density; Ov, overexpression plasmid; PCNA, proliferating cell nuclear antigen; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; TPX2, targeting protein for xenopus kinesin-like protein 2.

Fig 3: TPX2 interacts with NCOA5. (A) General biological repository for interaction data sets (https://bioplex.hms.harvard.edu/) was used to explore the BioPlex interaction data and identify high-confidence NCOA5 interactors. (B) Correlation analysis of TPX2 and NCOA5 (Gene Expression Profiling Interactive Analysis). (C) Co-immunoprecipitation assay for verification of the interaction between TPX2 and NCOA5. (D) TPX2 expression in paired non-tumor and tumor tissues of patients with breast cancer was analyzed using TNMplot tool. (E) MCF7 cells were transfected with si-TPX2 or si-NC. The transfection efficiency of si-TPX2 in MCF7 cells was validated via RT-qPCR. (F) MCF7 cells were transfected with si-TPX2 or si-NC. RT-qPCR for determination of NCOA5 mRNA levels in MCF7 cells. (G) MCF7 cells were transfected with si-TPX2 or si-NC. Western blot analysis for determination of NCOA5 protein expression in MCF7 cells. **P<0.01, ***P<0.001. NC, negative control; NCOA5, nuclear receptor coactivator 5; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; TPM, transcript per million; TPX2, targeting protein for xenopus kinesin-like protein 2.

Fig 4: TPX2 knockdown induces weaker in vitro angiogenesis by downregulating NCOA5. CM of MCF7 cells after transfection with si-TPX2 or co-transfection with si-TPX2 and Ov-NCOA5 were collected and HUVECs were incubated with CM at 37°C for 24 h. (A) Tube formation assay for determination of in vitro angiogenesis of HUVECs. Scale bar, 250 µm. (B) Western blot analysis for determination of VEGFA and VEGFR2 protein expression in HUVECs. *P<0.05, ***P<0.001. CM, conditioned media; HUVECs, human immortalized umbilical vein endothelial cells; NC, negative control; NCOA5, nuclear receptor coactivator 5; Ov, overexpression plasmid; si, small interfering RNA; TPX2, targeting protein for xenopus kinesin-like protein 2.