Fig. 3

Functional characterization of combined EZH2/ATM inhibition. A, B Colony formation assay in BRCA1-deficient (KB1P-G3 and KB1P-B11) and BRCA1-proficient (KP-3.33 and KP-6.3) mouse mammary tumor cells after 7 days of incubation. A Representative microscopic images of colony formation at 0.65× magnification. Scale bar 200 µm. B Quantification of crystal violet positive stained cells is presented as mean ± SD of six independent experiments. Calculation of drug synergy using the Bliss independence score revealed a mean synergy score of 55.5 ± 9.8% in KB1P-G3, 50.7 ± 21.9% in KB1P-B11, −1.6 ± 20.2% in KP-3.33, and −1.3 ± 18.4% in KP-6.3 cells. C Growth inhibition curves using IncuCyte live cell imaging measured by cell densities on culture plates from at least three independent experiments with each three technical replicates. Bliss synergy score: KB1P-G3: 52.0 ± 8.1%; KB1P-B11: 32.7 ± 13.3%; KP-3.33: 2.4 ± 8.5%; KP-6.3: 3.7 ± 3.3%. D Flow cytometric analysis of apoptotic cells measured by Annexin V and propidium iodide double staining of cells treated with GSK126 and AZD1390 for 48 h. Bliss synergy score: KB1P-G3: 20.8 ± 10.3%; KB1P-B11: 22.4 ± 8.3%; KP-3.33: −4.5 ± 2.4%; KP-6.3: −0.4 ± 1.8%. All experiments were performed using inhibitor concentrations of 7.5 µM GSK126 and 2 µM AZD1390. Significance was tested by one-way ANOVA with Tukey multiple comparison test. CFU colony forming unit