Fig 2: Ovarian cancer cell growth in culture with and without CFI-400945 PLK4 inhibitor: KOC-7C compared with OVCAR3. (A) Cell proliferation for KOC-7C ovarian cancer cells without (DMSO) and with CFI-400945 PLK4 inhibitor (CFI-945) showing modest reduction in cell proliferation. N at hour 0 = 1 × 106 cells. (B) Cell proliferation of OVCAR3 ovarian cancer cells without (DMSO) and with CFI-400945 PLK4 inhibitor (CFI-945) showing substantial inhibition of cell proliferation. N at hour 0 = 1 × 106 cells. (C) Flow cytometry of control KOC-7C ovarian cancer cells after 24 h in culture (DMSO). (D) Flow cytometry of control OVCAR3 ovarian cancer cells after 24 h in culture (DMSO). (E) Flow cytometry of CFI-400945 inhibitor (50 nM)-treated OVCAR3 ovarian carcinoma cells after 24 h, with large residual population of cells retaining baseline modal DNA index (blue arrow), with only partial response to inhibitor. (F) Flow cytometry of CFI-400945 inhibitor (50 nM)-treated OVCAR3 ovarian cancer cells demonstrating no residual population of cells retaining the baseline modal DNA index (red arrow), indicating a complete response to inhibitor. Of note, the differential response observed here is not the result of variations in the population doubling time of the cell lines (KOC-7C = 21.00 h, OVCAR-3 = 19.49 h). Movies S1 and S2 show videomicroscopy of OVCAR3 cells with and without PLK4 CFI-400945 inhibitor.

Fig 3: Localization of phospho-PLK4 to centrosomes, kinetochores, and midbody varies with the phase of the cell cycle when an antibody to the N-terminal PEST domain is used. Phospho-serine305-PLK4 is identified first in centrosomes of interphase and prophase cells followed by kinetochores (metaphase and anaphase) and, subsequently, the cleavage furrow (telophase) and midbody (cytokinesis) of synchronized cells by immunofluorescence (IF) microscopy using an anti–phospho-serine305-PLK4 rabbit polyclonal antibody (our Ab#3 to a PLK4-serine305 peptide; red IF). Anti–a-tubulin antibody (green IF) identifies centrosomes, spindle apparatus, and midbody. DAPI stain (blue) identifies DNA. Results are illustrated for OVCAR3 ovarian cancer cells photographed at 400× original magnification. (Lower) Schematic diagram illustrates approximate location (arrows) of our rabbit polyclonal antibody (Ab#3, anti-phospho-PLK4) for phospho-PLK4. Similar results were obtained with a second, independently produced anti–phospho-serine305-PLK4 antibody (14299) from the laboratory of G.P.N.

Fig 4: Immunofluorescence localization of PLK4 to centrosomes, cleavage furrow, and midbody is altered compared with control (A, DMSO) by treatment with MG-115 protease inhibitor (A, MG115), CFI-400945 PLK4 kinase inhibitor (B, CFI-945), or both (B, CFI-945/MG115). Identification of PLK4 localization to centrosomes (A, DMSO, arrow) is improved by higher magnification (C) to illustrate size difference between centrosomes (arrows) and (substantially larger) midbodies. (D) PLK4 localization to midbodies is confirmed by colocalization with mitotic kinesin-like protein-1 (MKLP-1), a midbody protein. (E) Relative distribution of PLK4 to centrosomes and midbodies with DMSO, MG115, CFI-945, or MG115 and CFI945 treatment. Additional descriptive details are provided in the legend to SI Appendix, Fig. S4.

Fig 5: Western immunoblot of 19 different ovarian cell lines and one colorectal (NCI-H747) cancer cell line demonstrates full-length PLK4 (Upper Left and Upper Right) and phospho-PLK4 (Lower Left and Lower Right). (Upper) Western blots demonstrate full-length (97-kDa) PLK4 with a commercially available anti-PLK4 antibody (P005; Cell Signaling 3258, recognizing an epitope centered on the cysteine458 amino acid). (Lower) Western blots were performed by using the same filter after washing with buffer and reprobing with an anti–phospho-serine305-PLK4 antibody (Ab#14299). Please note that the phospho-PLK4 in the lower filter is of substantially smaller size, ~60 kDa. The difference is appreciated as the full-length signals were not completely removed by stripping in the lower filters. Cultured cells were not synchronized with regard to cell cycle. We confirmed that the 60-kDa band corresponded to pPLK4 by removing this band from the gel and performing amino acid sequence analyses. We also confirmed that a second anti–phospho-PLK4 antibody (Ab#3) from our laboratory recognized phospho-PLK4 by using immunoprecipitation assays followed by PAGE with silver staining to identify the band recognized on Western blot and, finally, MS to assess the amino-acid sequence of protein band (amino-acid sequence provided in Results). SI Appendix, Fig. S1 shows results in 23 additional colorectal and 9 breast cancer cell lines. (Lower) Schematic illustration of PLK4 amino-acid sequence shows the relative locations of epitopes recognized by the two antibodies used for Western immunoblot analyses (Upper), one from the laboratory of G.P.N. recognizing phospho-serine305-PLK4, produced in collaboration with Cell Signaling Technology, and the second commercially available recognizing cysteine458-PLK4.