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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Nat Commun. 2014;5:3361. doi: 10.1038/ncomms4361

Figure 3. Loss of PTEN Reverses HRD and Confers PARP Inhibitor Resistance to BRCA1-Depleted Cells through Over-expression of TTK.

Figure 3

(a) MCF-10A cells were infected by the indicated lentiviral particles targeting the indicated genes. Control cells and knockdown cells were subjected to microarray analysis. The presence of the HRD gene signature was analyzed by supervised clustering analysis.

(b) Modified HR repair assay was performed in MCF-10A cells by transfecting cells with DRGFP DSB substrate plasmid and I-SceI plasmid through electroporation, followed by analysis of GFP-positive cells by flow cytometry 48 to 72 hours later. Student’s t-test was performed from results of three independent experiments as mean + SD.

(c) The rate of cell survival in response to olaparib was determined by colony formation assay. Each value was relative to control cells without treatment and represents the mean ± SD from three independent experiments. Student’s t-test showed that treatment response differed between BRCA1-PTEN double knockdown cells and single knockdown cells (P<0.001).

(d) Heat map of the HRD gene signature with the 26 genes most significantly changed in BRCA1-PTEN double knockdown cells compared to single-knockdown-cells.

(e) Microarray data from 295 breast cancers were clustered into basal-like, Her2-positive (Her2), luminal A, luminal B, and normal breast-like. Gene expression levels of TTK among the different breast cancer subtypes are indicated.

(f) Quantitative analysis of HR repair assay in cells transfected with TTK plasmids. Results are shown as mean + SD from three independent experiments. Student’s t-test showed that over-expression of TTK significantly increased HR repair efficiency (P<0.05). BRCA1 SMARTpool siRNA was used as a positive control. Western blots demonstrating effective knockdown are shown to the bottom.