br To investigate whether these effects

To investigate whether these effects are cell type-specific, we studied a second EOC cell line, SKOV-3. The treatment with increasing concentrations of paclitaxel reduced cellular viability and revealed an IC50 of 7 nM (Supplementary Figure S2A). For BI6727 an IC50 of 28 nM, for paclitaxel/BI6727 an IC50 of 2.4 nM, and for proTAME an IC50 of 12.5 μM were determined (Supplementary Figure S2, B-D). The sequential triple combination with increasing concentrations of paclitaxel and 20 nM BI6727 for 24 hours followed by 10 μM pro TAME for additional 24 hours reduced the IC50 for paclitaxel to 1.4 nM (Supplementary Figure S2E), supporting that the combinatorial treatment with a PLK1 inhibitor and proTAME reduces significantly the IC50 of paclitaxel in ovarian cancer cells.
The Hexa His peptide analysis using a flow cytometer (FACS) revealed that 2.5 nM paclitaxel (24 hours) induced a small increase of OVCAR-3 cells in the G2/M phase from 19% to 21% due to prolonged mitotic arrest as shown by microscopical inspection. The incubation with 15 nM BI6727 (24 hours) increased the fraction of cells in the G2/M phase to 44% and the treatment with 10 μM proTAME to 26% (Supplementary Figure S3A). The combinatorial treatment in OVCAR-3 and SKOV-3 cells showed the most pronounced enrichment of cells in G2/M with 62% and 78%, respectively (Supplementary Figure S3, A and B).
Blocking the APC/C Inhibits Long-Term Growth of Mitotically Arrested Ovarian Cancer Cells
To assess whether the APC/C could be a potential target to inhibit the long-term growth of ovarian cancer cells arrested in mitosis, we tested first the activity of proTAME in OVCAR-3 cells. In a co-immunoprecipitation experiment with increasing concentrations of proTAME, we could demonstrate the reduced association of CDC20 and a component of the APC/C, CDC27, in a dose-dependent manner (Figure 2A). Inhibiting the binding of CDC20 to the CDC27 prevents activation of the APC/C stabilizing Cyclin B1 (Figure 2A, upper panel), which plays an important role to keep cells arrested in mitosis.
Despite a strong impact of the triple combination on the viability after 48 hours of treatment, we determined the percentage of remaining,
still viable cells to be 20% for OVCAR-3 cells and 30% for SKOV-3 cells, respectively (Supplementary Figures S1E and S2E). Therefore, we conducted experiments to investigate the fate of ovarian cancer cells over a prolonged period of time. We inhibited the mitotic exit of paclitaxel/ BI6727-treated cells by incubation with proTAME and tested the 
viability up to 96 hours. While single agents at concentrations of 2.5 nM paclitaxel, 10 nM BI6727, or 10 μM proTAME could not prevent an increase in cell numbers, only the combinations paclitaxel/ BI6727 and paclitaxel/BI6727/proTAME reduced cell numbers significantly (P b .001) (Figure 2B). We observed the most pronounced
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Figure 2. Combinatorial treatment of mitotic OVCAR-3 cells with the APC/C inhibitor proTAME induces a long-lasting inhibition of cell growth. (A) OVCAR-3 cells were treated for 24 hours with paclitaxel and BI6727 followed by a 6-hour treatment with proTAME. Whole cell lysates were used for co-immunoprecipitation experiments with CDC27 antibodies followed by western blotting using antibodies (lower panel) for CDC20 and (upper panel) for Cyclin B1, CDC27, and β-Actin. (B) OVCAR-3 cells were treated for up to 96 hours with 2.5 nM paclitaxel (Pac), 10 nM BI6727, 10 μM proTAME, 2.5 nM Pac/10 nM BI6727, or 2.5 nM Pac/10 nM BI6727/10 μM proTAME for the determination of cell viability (*P ≤ .05; **P ≤ .01; ***P ≤ .001). (C) Coomassie-stained regrown colonies of OVCAR-3 cells treated with 2.5 nM paclitaxel, 10 nM BI6727, 20 μM proTAME, or combinations thereof. The number of colonies was determined after 21 days. Numbers were statistically significant by two-tailed Student's t test (**P ≤ .01). Each bar graph represents the mean value ± SEM (n=3).
(D) OVCAR-3 cells were grown as 3D culture over 14 days and treated with 10 nM BI6727, 2.5 nM Pac, and/or 20 μM proTAME. Cells were stained using the LIVE/DEAD viability/cytotoxicity kit, and ratios of viable/dead cells were calculated. Measurements were statistically significant by two-tailed Student's t test (*P ≤ .05). Each bar graph represents the mean value ± SEM (n=3).
decrease in the viability of mitotic OVCAR-3 cells of treatment with paclitaxel/BI6727/proTAME, which left only 3% of ovarian cancer cells after 96 hours (Figure 2B).