Figure S3.
Analysis of Nek2A overexpression and DAs.(A) Analysis of Nek2A overexpression. Representative images of RPE1 cells carrying either mNeonGreen-Nek2A WT or mNeonGreen-Nek2A-KD under control of the DOX-inducible promoter. Cells were treated with solvent only (− DOX) or DOX (+ DOX) and analyzed by immunofluorescence using γ-tubulin (γ-tub; as a centrosome marker) and direct mNeonGreen fluorescence to detect Nek2. DAPI stained the DNA. Scale bar, 20 µm. Graphs show the quantification of Nek2A-WT– and Nek2A–KD–overexpressing cells. Bar graphs represent mean ± SD. Only cells treated with DOX were analyzed. n = 335 for mNeonGreen-Nek2A (Nek2-WT) and n = 424 for mNeonGreen-Nek2A-KD RPE1 cells in three independent experiments. Western blot analysis of RPE1 cells carrying either mNeonGreen-Nek2A WT or mNeonGreen-Nek2A-KD under control of the DOX-inducible promoter treated with solvent (− DOX) or DOX (+ DOX) and stained with anti-Nek2 antibodies. The upper band corresponds to mNeonGreen-Nek2A, and the lower and weaker Nek2 band is the size of endogenous Nek2. Actin was used as a loading control. (B) Box/dot plots show quantifications of the fluorescence intensity of the indicated proteins upon C-Nap1 siRNA treatment and DOX induction of mNeonGreen-Nek2A (Nek2-WT). Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. Data from three independent experiments are shown. n = 150 cells per condition. (C) Proteasome inhibition increases the levels of Nek2 at centrosomes. RPE1 cells carrying mNeonGreen-Nek2A were treated with DOX as described in A in the absence or presence of the proteasome inhibitor MG132 (MG). Cells were subjected to immunofluorescence analysis using anti–γ-tubulin (γ-tub) antibodies (centrosome marker). mNeonGreen-Nek2A was visualized by direct fluorescence. DAPI stained the DNA. The boxplot shows the fluorescence intensity in a.u. of mNeonGreen-Nek2A. Boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. n = 50 cells per condition. As published (Spalluto et al., 2012), Nek2 accumulates at the centrosome after proteasome inhibition. Scale bars, 20 µm (panel) and 2 µm (inset). (D) RPE1 cells were treated with solvent (− MG132 [MG]) or MG132 and analyzed by immunofluorescence using anti-Nek2 and anti–γ-tubulin (γ-tub) antibodies. The boxplot shows the fluorescence intensity in a.u. of Nek2 at centrosomes. Boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. n = 50 cells per condition. (E) Western blot analysis of RPE1 cells in interphase (Int) and mitosis (Mit). Mitotic cells were enriched by mitotic shake off. Cells were lysed and analyzed with the indicated antibodies. The appearance of slower migrating forms of GM130 as well as Histone H3 serve as a marker for mitotic cells. Actin was used as a loading control. (F) Quantification of Nek2 knockdown in MCF10A, MCF10AT, and MCF10CA1 cells. The indicated cell lines were treated with control (siCtrl) or Nek2 siRNA and Nek2 centrosomal levels were analyzed by immunofluorescence using anti-Nek2 and γ-tubulin antibodies. The graph shows the percentage of Nek2-positive interphase cells for each condition. Bar graphs represent mean ± SD. n > 450 cells were analyzed per cell type and treatment in a total of four independent experiments (three in the case of MCF10CA1). (G) RPE1 expressing mNeonGreen-Nek2A under control of the DOX-inducible promoter were treated with solvent only (− DOX) or DOX (+ DOX) in the presence of absence of the Plk1 inhibitor BI-2536 for 1 or 6 h. Cells were analyzed by immunofluorescence using anti-Cep164 and γ-tubulin antibodies. γ-tubulin serves as a centrosome marker. The DNA was stained with DAPI. Boxplots show the quantification of Cep164 signal at the centrosome. Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. Data from one experiment are shown. n = 150 cells per experimental condition. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Analysis of Nek2A overexpression and DAs. (A) Analysis of Nek2A overexpression. Representative images of RPE1 cells carrying either mNeonGreen-Nek2A WT or mNeonGreen-Nek2A-KD under control of the DOX-inducible promoter. Cells were treated with solvent only (− DOX) or DOX (+ DOX) and analyzed by immunofluorescence using γ-tubulin (γ-tub; as a centrosome marker) and direct mNeonGreen fluorescence to detect Nek2. DAPI stained the DNA. Scale bar, 20 µm. Graphs show the quantification of Nek2A-WT– and Nek2A–KD–overexpressing cells. Bar graphs represent mean ± SD. Only cells treated with DOX were analyzed. n = 335 for mNeonGreen-Nek2A (Nek2-WT) and n = 424 for mNeonGreen-Nek2A-KD RPE1 cells in three independent experiments. Western blot analysis of RPE1 cells carrying either mNeonGreen-Nek2A WT or mNeonGreen-Nek2A-KD under control of the DOX-inducible promoter treated with solvent (− DOX) or DOX (+ DOX) and stained with anti-Nek2 antibodies. The upper band corresponds to mNeonGreen-Nek2A, and the lower and weaker Nek2 band is the size of endogenous Nek2. Actin was used as a loading control. (B) Box/dot plots show quantifications of the fluorescence intensity of the indicated proteins upon C-Nap1 siRNA treatment and DOX induction of mNeonGreen-Nek2A (Nek2-WT). Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. Data from three independent experiments are shown. n = 150 cells per condition. (C) Proteasome inhibition increases the levels of Nek2 at centrosomes. RPE1 cells carrying mNeonGreen-Nek2A were treated with DOX as described in A in the absence or presence of the proteasome inhibitor MG132 (MG). Cells were subjected to immunofluorescence analysis using anti–γ-tubulin (γ-tub) antibodies (centrosome marker). mNeonGreen-Nek2A was visualized by direct fluorescence. DAPI stained the DNA. The boxplot shows the fluorescence intensity in a.u. of mNeonGreen-Nek2A. Boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. n = 50 cells per condition. As published (Spalluto et al., 2012), Nek2 accumulates at the centrosome after proteasome inhibition. Scale bars, 20 µm (panel) and 2 µm (inset). (D) RPE1 cells were treated with solvent (− MG132 [MG]) or MG132 and analyzed by immunofluorescence using anti-Nek2 and anti–γ-tubulin (γ-tub) antibodies. The boxplot shows the fluorescence intensity in a.u. of Nek2 at centrosomes. Boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. n = 50 cells per condition. (E) Western blot analysis of RPE1 cells in interphase (Int) and mitosis (Mit). Mitotic cells were enriched by mitotic shake off. Cells were lysed and analyzed with the indicated antibodies. The appearance of slower migrating forms of GM130 as well as Histone H3 serve as a marker for mitotic cells. Actin was used as a loading control. (F) Quantification of Nek2 knockdown in MCF10A, MCF10AT, and MCF10CA1 cells. The indicated cell lines were treated with control (siCtrl) or Nek2 siRNA and Nek2 centrosomal levels were analyzed by immunofluorescence using anti-Nek2 and γ-tubulin antibodies. The graph shows the percentage of Nek2-positive interphase cells for each condition. Bar graphs represent mean ± SD. n > 450 cells were analyzed per cell type and treatment in a total of four independent experiments (three in the case of MCF10CA1). (G) RPE1 expressing mNeonGreen-Nek2A under control of the DOX-inducible promoter were treated with solvent only (− DOX) or DOX (+ DOX) in the presence of absence of the Plk1 inhibitor BI-2536 for 1 or 6 h. Cells were analyzed by immunofluorescence using anti-Cep164 and γ-tubulin antibodies. γ-tubulin serves as a centrosome marker. The DNA was stained with DAPI. Boxplots show the quantification of Cep164 signal at the centrosome. Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. Data from one experiment are shown. n = 150 cells per experimental condition. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

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