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  • br Time Lapse Microscopy br

    2020-08-18


    Time-Lapse Microscopy
    Thymidine-synchronized ovarian cells expressing mCherry-histone H2B were released for 5 hours, treated either with single agents or combinations. For time-lapse analysis, the treated cells were transferred to the microscope stage, and microscopy was performed with Axioimager inverted Z1 (Zeiss) equipped with an environmental chamber (Zeiss) that maintained the cells at 37°C in a humidified environment of 5% CO2. Images were taken every 10 minutes using an Axiocam MRm camera (Zeiss) driven by Axiovision SE64 software (Zeiss). Movies and JPEG files were imported into ImageJ and proceeded using the same software. Nuclear envelope breakdown was judged as such when the nuclear membrane lost a smooth and the linear periphery. The first frame showing a poleward movement of the chromosomes was defined as Trametinib onset.
    Chromosome Spreads
    Cells were treated overnight with 3.3 μM Nocodazol. The next day, cells were harvested by mitotic shake off and hypotonically swollen in 40% medium/ 60% tap water for 20 minutes at 37°C. Cells were fixed with freshly made Carnoy's solution (75% methanol, 25% acetic acid), and the fixative was changed several times. For spreading, cells in Carnoy's solution were dropped onto prechilled glass slides. Slides were dried at room temperature for 24 hours and stained with DAPI. Chromosome number per condition was counted using an AxioObserver.Z1 microscope with a HCX PL APO CS 63.0x1.4 oil UV objective (Zeiss, Göttingen). The graphic representation of the results was done using GraphPad Prism software.
    Cell Proliferation and Caspase-3/7 Activity (Multiplexed Protocol) Statistical Analysis
    Forty-eight hours following transfection, 7 μl substrate of the Cell Titer-Blue Cell Viability Assay (Promega) was added to each well. After a short centrifugation step (1000 rpm for 10 seconds), cells were incubated  All experiments were performed at least three times and displayed as mean and standard error of the mean. The statistical significance was assessed by Student's t test (two-tailed and paired) using Excel
    Image Work
    Images were opened in Adobe Photoshop CS6, sized, and placed in figures using Adobe Illustrator CS6 (Adobe Systems, Mountain View, CA).
    Results
    PLK1 Gene Expression and Survival of Ovarian Cancer Patients
    At first, we studied the prognostic role of PLK1 expression in ovarian cancer patients and evaluated the correlation between PLK1 expression and patient's survival based on methods for survival analysis. One hundred sixteen patients (44.1%) had high PLK1 expression, and 147 patients (55.8%) displayed low PLK1 detection. According to a Kaplan-Meier analysis, patients in clinical stages I and II with a high PLK1 (WSN 6) expression displayed a significantly (P = .028) impaired overall survival (62.3 months, 95% confidence interval: 52.8-76.8) compared to those having a low PLK1 expression (75.9 months, 95% confidence interval: 68.1-83.7) (Figure 1A).
    Small Molecule APC/C Inhibitors Decrease the Viability of Mitotically Arrested Ovarian Cancer Cells and Sensitize Cells to Paclitaxel
    Considering the prognostic and potential therapeutic role of PLK1 for early- and late-stage ovarian cancer, we determined the viability of
    A
    B
    PLK1 high
    survival
    PLK1 low
    Overall
    PLK1 low
    PLK1 high
    Time (months)
    primarytumor(relapse)
    kDa OVCAR-3SKOV-3 primarytumor
    C
    PLK1
    -Actin
    Figure 1. Kaplan-Meier analysis of ovarian cancer patients in stages I/II and examination of PLK1 expression. (A) Overall survival in 263 patients with ovarian cancer stage I and II disease according to PLK1 expression based on immunohistochemical evaluation of tumor resection specimens. (B) Representative examples of ovarian carcino-ma with a low (WS ≤ 6) and high (WSN 6) detection of PLK1 in tumor cells. Original magnification × 10, scale bars 250 μm. (B) Whole cell lysates of OVCAR-3, SKOV-3, and primary ovarian cancer cells were analyzed for PLK1 expression. Endogenous levels of PLK1 and β-Actin were determined by immunoblotting. 
    OVCAR-3 cells expressing PLK1 (Figure 1B) upon treatment with the potent clinical PLK inhibitor BI6727 [47] or paclitaxel. The treatment with increasing concentrations revealed IC50s of 2.75 nM for paclitaxel and 15 nM for BI6727, respectively (Supplementary Figure S1, A and B). In a combinatorial approach including 10 nM BI6727, the IC50 for paclitaxel dropped to 1.4 nM (Supplementary Figure S1C).
    The antiproliferative basis for paclitaxel has long been thought to be due to the presence of an activated SAC that culminates in a mitotic cell death [40]. However, depending on the biological context and cell type, cells can escape from cell death by mitotic slippage. Thus, mitotic slippage represents a potential mechanism limiting the effectiveness of paclitaxel. To elucidate the importance and the therapeutic potential of an APC/C inhibitor that blocks exit of mitotically arrested cells induced by paclitaxel/BI6727, we tested first the effect of increasing proTAME concentrations on OVCAR-3 cells and determined the IC50 to be 12.5 μM (Supplementary Figure S1D). In addition, cells were pretreated for 24 hours with increasing concentrations of paclitaxel and 10 nM BI6727 to induce a strong mitotic arrest followed by 20 μM proTAME for additional 24 hours. The treatment with BI6727 and proTAME decreased the IC50 of paclitaxel further to 0.9 nM (Supplementary Figure S1E). In all, following combinatorial experiments including the mitotic inhibitors (paclitaxel, BI6727) and proTAME, we treated cells first with mitotic inhibitor/s for 24 hours followed by an incubation period of 24 hours with proTAME.