PLK

Western blot analysis. HeLa or MCF7 cells were treated with nocodazole (noc, 50 ng/ml), paclitaxel (pacli, 50 nM), the Plk1 inhibitor BI 2536 (50 nM) or BI 6727 (50 nM) for 16 h and cellular extracts were prepared for western blot analysis with antibodies as indicated. con: cellular extracts from control cells without any treatment. β-actin served as loading control.

L3.6pl cells at 6,000 cells per well were incubated in MEM with 5% FBS in triplicate in a 96-well culture plate and then treated alone with 5 umol/L BMS-777607, 10 umol/L wortmannin, or with BMS-777607 in combination with individual inhibitors. Polyploidy was examined under BK71 Olympus microscope and photographed 72 hours after treatment.

Along with cell death, immunoblotting shows ON 01910.Na induces hyperphosphorylation of RanGAP1, increased expression of RanGAP1.SUMO1 but decreased expression of free unmodified RanGAP1. No viable SU-DHL-5 cells were available for immunoblotting at 0.5 uM of ON 01910.Na.

Cells with decreased GRK5 expression are more sensitive to PLK1 inhibition.A , shown is cell death in control or GRK5-shRNA stably transfected cells after 72 h of treatment with 0-120 nM BI 2536 as determined by trypan blue exclusion (n = 6).B , shown is cell death in control or GRK5-shRNA stably transfected cells after 72 h treatment with 0 –120 nM GSK461364, as determined by trypan blue exclusion ( n = 4).

Intracellular trafficking of plasmid DNA in the presence and absence of PLK1 inhibitors. PC3-PSMA cells were transfected with polyplexes of PEI:fluorescein (green)-labeled plasmid DNA at a weight ratio of 1:4. Cells treated with A) vehicle control (DMSO) and B) 25 nM BI 2536 are shown 48 h after co-treatment with polyplexes. Cells treated with C) vehicle control (DMSO) and D) HMN-214 are shown 24 h after co-treatment with polyplexes. Scale bar = 20 μm. Blue signal indicates DAPI-stained nuclei, and white arrows indicate sequestration of plasmid DNA in the perinuclear recycling compartment (PNRC). Red arrows indicate altered plasmid DNA localization following treatment with PLK1 inhibitors. Images are representative of three independent experiments (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

RO3280 promotes the formation of multinucleated cells in bladder cancer cell lines. (A) Representative images of cells stained with DAPI (blue) and alpha‐tubulin (red) were examined under a fluorescence microscope (magnification 400×). Enlarged cells containing multiple evenly stained nuclei (multinucleated cells) are characteristic of mitotic catastrophe (white arrow). (B) Quantification of the percentage of multinucleated cells based on fluorescence microscopy analysis (the data are presented as the mean ± S.E.M., *P < 0.05).

Western blot analysis. HeLa or MCF7 cells were treated with nocodazole (noc, 50 ng/ml), paclitaxel (pacli, 50 nM), the Plk1 inhibitor BI 2536 (50 nM) or BI 6727 (50 nM) for 16 h and cellular extracts were prepared for western blot analysis with antibodies as indicated. con: cellular extracts from control cells without any treatment. β-actin served as loading control.

L3.6pl cells at 6,000 cells per well were incubated in MEM with 5% FBS in triplicate in a 96-well culture plate and then treated alone with 5 umol/L BMS-777607, 10 umol/L wortmannin, or with BMS-777607 in combination with individual inhibitors. Polyploidy was examined under BK71 Olympus microscope and photographed 72 hours after treatment.

Along with cell death, immunoblotting shows ON 01910.Na induces hyperphosphorylation of RanGAP1, increased expression of RanGAP1.SUMO1 but decreased expression of free unmodified RanGAP1. No viable SU-DHL-5 cells were available for immunoblotting at 0.5 uM of ON 01910.Na.

Cells with decreased GRK5 expression are more sensitive to PLK1 inhibition.A , shown is cell death in control or GRK5-shRNA stably transfected cells after 72 h of treatment with 0-120 nM BI 2536 as determined by trypan blue exclusion (n = 6).B , shown is cell death in control or GRK5-shRNA stably transfected cells after 72 h treatment with 0 –120 nM GSK461364, as determined by trypan blue exclusion ( n = 4).

Intracellular trafficking of plasmid DNA in the presence and absence of PLK1 inhibitors. PC3-PSMA cells were transfected with polyplexes of PEI:fluorescein (green)-labeled plasmid DNA at a weight ratio of 1:4. Cells treated with A) vehicle control (DMSO) and B) 25 nM BI 2536 are shown 48 h after co-treatment with polyplexes. Cells treated with C) vehicle control (DMSO) and D) HMN-214 are shown 24 h after co-treatment with polyplexes. Scale bar = 20 μm. Blue signal indicates DAPI-stained nuclei, and white arrows indicate sequestration of plasmid DNA in the perinuclear recycling compartment (PNRC). Red arrows indicate altered plasmid DNA localization following treatment with PLK1 inhibitors. Images are representative of three independent experiments (N = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

RO3280 promotes the formation of multinucleated cells in bladder cancer cell lines. (A) Representative images of cells stained with DAPI (blue) and alpha‐tubulin (red) were examined under a fluorescence microscope (magnification 400×). Enlarged cells containing multiple evenly stained nuclei (multinucleated cells) are characteristic of mitotic catastrophe (white arrow). (B) Quantification of the percentage of multinucleated cells based on fluorescence microscopy analysis (the data are presented as the mean ± S.E.M., *P < 0.05).