![Figure 3. Nek5 loss leads to increased rootletin but reduced PCM at interphase centrosomes. (a) Staining of γ-tubulin together with Nek5, C-Nap1, rootletin, or Nek2 in U2OS cells treated with siRNAs against GL2 or Nek5 for 48 h. (b) Histogram of relative intensities of Nek5, γ-tubulin, C-Nap1, rootletin, and Nek2 in U2OS cells mock-treated or with siRNAs against GL2 or Nek5 for 48 h. (c) Histogram of relative intensities of γ-tubulin, C-Nap1, rootletin, and Nek2 in U2OS cells transfected with GFP, GFP-Nek5WT, or GFP-Nek5KD for 48 h. Representative images from GFP-Nek5WT– and GFP-Nek5KD–transfected cells stained for each protein are shown. (d) Staining of rootletin and γ-tubulin in mock- and Nek5-depleted cells. (e) Staining of GFP and γ-tubulin in cells transfected with GFP-rootletin for 24 h. (f) Quantification of cells with centrosomes separated by >2 µm in mock-, GFP-, or GFP-rootletin–transfected cells. (g) Staining of rootletin and γ-tubulin in mock-, Nek2-, or Nek2- and Nek5-depleted cells. (h) Western blot analysis of C-Nap1, rootletin, Nek2, and α-tubulin from mock-, GL2-, Nek5- or Nek2-depleted cells. (i) Quantification of cells with centrosomes separated by >2 µm in mock-, GL2-, Nek5-, Nek2-, or Nek2- and Nek5-depleted U2OS cells. (j) Quantification of cells with centrosomes separated by >2 µm in untransfected or GFP-Nek2KD–transfected mock-, GL2-, or Nek5-depleted U2OS cells. (k) Staining of centrin, Cdk5Rap2, Cep192, and pericentrin in GL2- and Nek5-depleted cells. (l) Histogram of intensities of centrin, Cdk5Rap2, Cep192, and pericentrin in U2OS cells treated with siRNAs against GL2 or Nek5 for 48 h relative to mock-treated cells. (m) Staining of pericentrin, EB1, and α-tubulin in GL2- and Nek5-depleted cells treated with nocodazole for 5 h and 60 s washout to allow MT regrowth. (n) Histogram of the diameter of MT asters formed after 60 s regrowth in mock-, GL2-, and Nek5-depleted cells. Bars: (a, k, and m, main images) 10 µm; (a, k, and m [insets], c–e, and g) 1 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Histograms show mean + SD (error bars); n = 3, 40 centrosomes (b, c, l, and n) or 200 cells (f, i, and j) per experiment.](https://cdn.rupress.org/rup/content_public/journal/jcb/209/3/10.1083_jcb.201412099/7/jcb_201412099_fig3.jpeg?Expires=1771573775&Signature=1pKPAS0tzsbsAPEM4e3yQTOWOP47KUggkUH2oKbfCWg9NWZTT8GZvW4QFyL29Su0Zx9oOJbWD3bZAa7aU-2vAqTvwK-LC6Xb~wxzmvXrWSkeTXhfsO77YF39yrZD4PD2OeyORC9E3NVc7l~IatYG35yY2mdrX4o3lXqxx1SVYO5Es5zxwU2yr8YreCqC3nMsBKHzxP2eeHqcZJFKS8pMF1Ce3LbyzzvlgHd3g~gciUUatGbnBIgNnPu~ye1zjnzQjvx3DUNlotYpwPOOLW1C2VHf9MLuSVRk5v2ugYtlSXtDNdXDLfivNcq1m2lN50kR9xr0mNB5mQ1HKgAjyZO5fA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Nek5 loss leads to increased rootletin but reduced PCM at interphase centrosomes. (a) Staining of γ-tubulin together with Nek5, C-Nap1, rootletin, or Nek2 in U2OS cells treated with siRNAs against GL2 or Nek5 for 48 h. (b) Histogram of relative intensities of Nek5, γ-tubulin, C-Nap1, rootletin, and Nek2 in U2OS cells mock-treated or with siRNAs against GL2 or Nek5 for 48 h. (c) Histogram of relative intensities of γ-tubulin, C-Nap1, rootletin, and Nek2 in U2OS cells transfected with GFP, GFP-Nek5WT, or GFP-Nek5KD for 48 h. Representative images from GFP-Nek5WT– and GFP-Nek5KD–transfected cells stained for each protein are shown. (d) Staining of rootletin and γ-tubulin in mock- and Nek5-depleted cells. (e) Staining of GFP and γ-tubulin in cells transfected with GFP-rootletin for 24 h. (f) Quantification of cells with centrosomes separated by >2 µm in mock-, GFP-, or GFP-rootletin–transfected cells. (g) Staining of rootletin and γ-tubulin in mock-, Nek2-, or Nek2- and Nek5-depleted cells. (h) Western blot analysis of C-Nap1, rootletin, Nek2, and α-tubulin from mock-, GL2-, Nek5- or Nek2-depleted cells. (i) Quantification of cells with centrosomes separated by >2 µm in mock-, GL2-, Nek5-, Nek2-, or Nek2- and Nek5-depleted U2OS cells. (j) Quantification of cells with centrosomes separated by >2 µm in untransfected or GFP-Nek2KD–transfected mock-, GL2-, or Nek5-depleted U2OS cells. (k) Staining of centrin, Cdk5Rap2, Cep192, and pericentrin in GL2- and Nek5-depleted cells. (l) Histogram of intensities of centrin, Cdk5Rap2, Cep192, and pericentrin in U2OS cells treated with siRNAs against GL2 or Nek5 for 48 h relative to mock-treated cells. (m) Staining of pericentrin, EB1, and α-tubulin in GL2- and Nek5-depleted cells treated with nocodazole for 5 h and 60 s washout to allow MT regrowth. (n) Histogram of the diameter of MT asters formed after 60 s regrowth in mock-, GL2-, and Nek5-depleted cells. Bars: (a, k, and m, main images) 10 µm; (a, k, and m [insets], c–e, and g) 1 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Histograms show mean + SD (error bars); n = 3, 40 centrosomes (b, c, l, and n) or 200 cells (f, i, and j) per experiment.
