Included for analysis. Cells were scored as exhibiting “aligned” chromosomes if

Included for analysis. Cells were scored as exhibiting “aligned” chromosomes if all DNA, visualized by DAPI, was present at the cell equator. All other cells were scored as “misaligned”. For chromosome segregation, anaphase cells were identified by central spindle localization of GFP-Plk1as and chromosome position was determined by DNA and kinetochores signals. Cells were scored as exhibiting “segregated” chromosomes if no Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Lera et al. Page 13 lagging DNA or ACA signals PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985561 were observed. “Lagging” chromosomes were defined if single DNA or ACA signals were observed lagging behind the segregating masses or if the two masses were not sufficiently separated despite elongation of the cytoplasm. Quantitation of cytoplasmic 181223-80-3 fluorescence intensity was performed using Nikon Elements. Images of 10 cells were acquired for each condition per Plk1 construct. Threshold levels were equally applied to all images to exclude background intensity. Average volume intensity measurements for each channel were made using a 1.51.5 m box placed in the cytoplasm of each cell. To determine the quantity of construct extracted, the median intensity of the `with extraction’ cells was subtracted from individual intensities in the `without extraction’ cells. These values were then divided by the amount of GFP-Plk1as extracted to control for cell-cell variability. Sample size was selected for cell biology experiments based on prior experience and biologically significant effect size. For immunofluorescence, the sample size was typically ~100 cells. Three biologic replicates were performed each with this sample size. Data analysis was performed using Prism 6. Statistical significance was determined using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance when comparing multiple cell lines against the vector control or a two-tailed, unpaired t-test with Welch’s correction when comparing a single cell line with different chemical treatments. High Resolution Imaging and Delta analysis–Samples were prepared as previously 35 described. Image acquisition was performed on a Nikon TE300 inverted microscope equipped with a Yokogawa CSU10 spinning disk purchase MRT-67307 confocal with image magnification yielding a 65 nm pixel size from the Orca ER cooled CCD camera and an 100X/1.4NA DIC oil immersion objective. Sixty-five flame 3D stacks of pairs of red and green fluorescent images were obtained sequentially at 200 nm steps along the z-axis through the cell from coverslip surface using MetaMorph 6.1 software. For each metaphase kinetochore pair, 3D centroid positions were measured with a 3D 36 Gaussian fitting function as described previously. The centroids of one color were projected to the axis defined by the centroids of the other color, and the Delta was calculated to correct for chromatic aberration. Mean Delta values were corrected for tilt of the face of the kinetochore relative to the axis between sister kinetochores. Mass Spectrometry Cell Culture–EGFP-Plk1as RPE1 cell lines stably expressing Flag-Plk1wt, Ch-Plk1aa, Flag-Plk1C-Kif2c, Flag-Plk1C-H2B, or Flag-Plk1C-Dsn1 were split in half and cultured as above. At approximately 60% confluence, cells were synchronized in S-phase with 3 mM thymidine. After 24 h, cells were rinsed twice with Hank’s Balanced Salt Solution, then replenished with fresh media contain.Included for analysis. Cells were scored as exhibiting “aligned” chromosomes if all DNA, visualized by DAPI, was present at the cell equator. All other cells were scored as “misaligned”. For chromosome segregation, anaphase cells were identified by central spindle localization of GFP-Plk1as and chromosome position was determined by DNA and kinetochores signals. Cells were scored as exhibiting “segregated” chromosomes if no Nat Chem Biol. Author manuscript; available in PMC 2016 October 04. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Lera et al. Page 13 lagging DNA or ACA signals PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985561 were observed. “Lagging” chromosomes were defined if single DNA or ACA signals were observed lagging behind the segregating masses or if the two masses were not sufficiently separated despite elongation of the cytoplasm. Quantitation of cytoplasmic fluorescence intensity was performed using Nikon Elements. Images of 10 cells were acquired for each condition per Plk1 construct. Threshold levels were equally applied to all images to exclude background intensity. Average volume intensity measurements for each channel were made using a 1.51.5 m box placed in the cytoplasm of each cell. To determine the quantity of construct extracted, the median intensity of the `with extraction’ cells was subtracted from individual intensities in the `without extraction’ cells. These values were then divided by the amount of GFP-Plk1as extracted to control for cell-cell variability. Sample size was selected for cell biology experiments based on prior experience and biologically significant effect size. For immunofluorescence, the sample size was typically ~100 cells. Three biologic replicates were performed each with this sample size. Data analysis was performed using Prism 6. Statistical significance was determined using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance when comparing multiple cell lines against the vector control or a two-tailed, unpaired t-test with Welch’s correction when comparing a single cell line with different chemical treatments. High Resolution Imaging and Delta analysis–Samples were prepared as previously 35 described. Image acquisition was performed on a Nikon TE300 inverted microscope equipped with a Yokogawa CSU10 spinning disk confocal with image magnification yielding a 65 nm pixel size from the Orca ER cooled CCD camera and an 100X/1.4NA DIC oil immersion objective. Sixty-five flame 3D stacks of pairs of red and green fluorescent images were obtained sequentially at 200 nm steps along the z-axis through the cell from coverslip surface using MetaMorph 6.1 software. For each metaphase kinetochore pair, 3D centroid positions were measured with a 3D 36 Gaussian fitting function as described previously. The centroids of one color were projected to the axis defined by the centroids of the other color, and the Delta was calculated to correct for chromatic aberration. Mean Delta values were corrected for tilt of the face of the kinetochore relative to the axis between sister kinetochores. Mass Spectrometry Cell Culture–EGFP-Plk1as RPE1 cell lines stably expressing Flag-Plk1wt, Ch-Plk1aa, Flag-Plk1C-Kif2c, Flag-Plk1C-H2B, or Flag-Plk1C-Dsn1 were split in half and cultured as above. At approximately 60% confluence, cells were synchronized in S-phase with 3 mM thymidine. After 24 h, cells were rinsed twice with Hank’s Balanced Salt Solution, then replenished with fresh media contain.