Figure S2.
Phenotypical rescue of Nek2 KO cells.(A) Analysis of Nek2 in Nek2 KO cells. Immunofluorescence and Western blot analysis of RPE1 WT and RPE1 Nek2 KO cells stained with anti-Nek2 antibodies. DNA was stained with DAPI, and γ-tubulin serves as a centrosome marker. Actin was used as a loading control. Scale bar, 20 µm. (B) Quantification of the FACS profile of RPE1 WT, Nek2 KO, and DOX-treated Nek2A-mNeonGreen–overexpressing cells stained with propidium iodide for DNA content analysis. Results represent mean ± SD from four independent experiments. (C) Western blot analysis of RPE1 WT and RPE1 Nek2 KO cells carrying mNeonGreen-Nek2A under control of the DOX-inducible promoter treated with 1 ng/ml DOX stained with anti-Nek2 antibodies. The lower Nek2 band (∼45 kD) is the size of endogenous Nek2, and the upper band corresponds to mNeonGreen-Nek2A. Actin was used as a loading control. (D) Quantification of cell cycle–dependent behavior of Cep164, Cep123, and LRRC45 in RPE1 Nek2 KO cells carrying mNeonGreen-Nek2A under control of the DOX-inducible promoter. Cells were incubated with solvent only (− DOX) or 1 ng/ml DOX (+ DOX) for 24 h and analyzed by immunofluorescence. Appendage proteins were labeled with specific antibodies. γ-tubulin serves as a centrosome marker. The levels of the indicated appendage protein were measured at each centrosome (centrosome 1 and centrosome 2) during interphase (inter), G2, prometaphase (pro), metaphase (meta), anaphase (ana), and telophase (telo) and normalized to the average of interphase (marked by gray shading). Graphs depict fluorescence average intensity in a.u. Bar graphs represent mean ± SD. N represents the total number of cells analyzed for each condition in three independent experiments. (E) Box/dot plots show quantifications of the fluorescence intensity of Cep164 in RPE1 Nek2 KO cells carrying mNeonGreen-Nek2A under control of the DOX-inducible promoter using solvent only (− DOX) and 1 ng DOX for 24 h (+ DOX). Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. N represents the total number of cells analyzed for each condition in three independent experiments. 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.

Phenotypical rescue of Nek2 KO cells. (A) Analysis of Nek2 in Nek2 KO cells. Immunofluorescence and Western blot analysis of RPE1 WT and RPE1 Nek2 KO cells stained with anti-Nek2 antibodies. DNA was stained with DAPI, and γ-tubulin serves as a centrosome marker. Actin was used as a loading control. Scale bar, 20 µm. (B) Quantification of the FACS profile of RPE1 WT, Nek2 KO, and DOX-treated Nek2A-mNeonGreen–overexpressing cells stained with propidium iodide for DNA content analysis. Results represent mean ± SD from four independent experiments. (C) Western blot analysis of RPE1 WT and RPE1 Nek2 KO cells carrying mNeonGreen-Nek2A under control of the DOX-inducible promoter treated with 1 ng/ml DOX stained with anti-Nek2 antibodies. The lower Nek2 band (∼45 kD) is the size of endogenous Nek2, and the upper band corresponds to mNeonGreen-Nek2A. Actin was used as a loading control. (D) Quantification of cell cycle–dependent behavior of Cep164, Cep123, and LRRC45 in RPE1 Nek2 KO cells carrying mNeonGreen-Nek2A under control of the DOX-inducible promoter. Cells were incubated with solvent only (− DOX) or 1 ng/ml DOX (+ DOX) for 24 h and analyzed by immunofluorescence. Appendage proteins were labeled with specific antibodies. γ-tubulin serves as a centrosome marker. The levels of the indicated appendage protein were measured at each centrosome (centrosome 1 and centrosome 2) during interphase (inter), G2, prometaphase (pro), metaphase (meta), anaphase (ana), and telophase (telo) and normalized to the average of interphase (marked by gray shading). Graphs depict fluorescence average intensity in a.u. Bar graphs represent mean ± SD. N represents the total number of cells analyzed for each condition in three independent experiments. (E) Box/dot plots show quantifications of the fluorescence intensity of Cep164 in RPE1 Nek2 KO cells carrying mNeonGreen-Nek2A under control of the DOX-inducible promoter using solvent only (− DOX) and 1 ng DOX for 24 h (+ DOX). Dots represent individual cells, boxes show interquartile range, lines inside the box represent the median, and whiskers show minimum and maximum values excluding outliers. N represents the total number of cells analyzed for each condition in three independent experiments. 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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