Figure 6. Supernumerary centrosomes have reduced PCM. (A) Representative TECs with the indicated centrosome numbers arrested in G1/S and stained for γ-tubulin, pericentrin, and DNA (DRAQ7). Boxes show areas of greater magnification. Numbered yellow lines, line scans of γ-tubulin intensity shown in graphs to the right. Bars, 5 µm. (B and C) Radial integrated protein intensities of γ-tubulin (B) or pericentrin (C) of TECs of the indicated centrosome numbers in control conditions (left) or after nocodazole (NOC) treatment (right). Scatter plot with mean (middle bars) and 95% confidence intervals are shown for each group (control: TEC 1–2, n = 52 centrosomes; TEC >2, n = 70 centrosomes; nocodazole treatment: TEC 1–2, n = 55 centrosomes; TEC >2, n = 57 centrosomes). Statistics: Student’s t test; ***, P < 0.001. (D) Diagram of setup that allowed centrosome or control-ablated cells to be relocated. (E) Radial integrated protein intensities of γ-tubulin between various groups. Scatter plot with mean (middle bars) and 95% confidence intervals are shown (one to two centrosomes, n = 26 centrosomes; noncentrosome ablation, n = 43 centrosomes; greater than two centrosomes, n = 29 centrosomes; after supernumerary centrosome ablation, n = 20 centrosomes). Statistics: Student’s t test; ***, P < 0.0001. (F) G1/S-arrested TECs with one to two (left) or greater than two (right) centrosomes in control conditions (top) or with BI2536 treatment and stained for γ-tubulin, pericentrin, and DNA (DRAQ7). Boxes show areas of greater magnification. Numbered yellow lines, line scans of γ-tubulin intensity graphed on the right. Bars: (main images) 5 µm; (insets) 2.5 µm. (G) Radial integrated densities of γ-tubulin in TECs of the indicated centrosome numbers in control conditions (left) or after Bl2536 treatment (right). Scatter plot with mean (middle bars) and 95% confidence intervals are shown (control: TEC 1–2, n = 28 centrosomes; TEC >2, n = 47 centrosomes; Bl2536 treatment: TEC 1–2, n = 63 centrosomes; TEC >2, n = 47 centrosomes). Statistics: Student’s t test; ***, P < 0.001. (H) G1/S-arrested TECs containing greater than two centrosomes in control conditions (left) or expressing Plk1::EGFP WT (right) and stained for (γ-tubulin) and DNA (DRAQ7). Numbered yellow lines, line scans of γ-tubulin intensity graphed to the right. Bars, 10 µm. (I) Mean γ-tubulin radial integrated protein density between the indicated groups normalized to controls. (TEC 1–2, n = 27 centrosomes; TEC >2, n = 27 centrosomes). Statistics: Student’s t test; error bars show means ± SEM; *, P < 0.05. (J) Mean centrosome perimeter between indicated groups. (TEC 1–2, n = 12 cells; TEC >2, n = 15 cells). Statistics: Student’s t test; error bars show means ± SEM; **, P < 0.01. (K) G1/S-arrested TECs containing greater than two centrosomes in control conditions (left) or expressing Plk1::EGFP USN (destruction box mutant, right) and stained for (γ-tubulin) and DNA (DRAQ7). Numbered yellow lines, line scans of γ-tubulin intensity graphed to the right. White asterisk denotes cluster of excess centrosomes. Bars, 10 µm. (L) Mean γ-tubulin radial integrated protein density between indicated groups normalized to controls. (TEC 1–2, n = 29 centrosomes; TEC >2, n = 25 centrosomes). Statistics: Student’s t test; error bars show means ± SEM; *, P < 0.05. (M) Mean centrosome perimeter between indicated groups. (TEC 1–2, n = 8 cells; TEC >2, n = 10 cells). Student’s t test; error bars show means ± SEM; **, P < 0.01. (N, left) Time-lapse fluorescence micrographs of G1/S-arrested TECs with one to two or greater than two centrosomes (cent) and expressing Plk1::EGFP WT (first and third rows), centrin::tdTomato (second row), or Plk1::EGFP USN (fourth row). Colored arrows, centrosomes. (right) Plots of centrosome perimeter over time. Bar, 5 µm. AU, arbitrary unit; Ctrl, control.
Figure 6.

Supernumerary centrosomes have reduced PCM. (A) Representative TECs with the indicated centrosome numbers arrested in G1/S and stained for γ-tubulin, pericentrin, and DNA (DRAQ7). Boxes show areas of greater magnification. Numbered yellow lines, line scans of γ-tubulin intensity shown in graphs to the right. Bars, 5 µm. (B and C) Radial integrated protein intensities of γ-tubulin (B) or pericentrin (C) of TECs of the indicated centrosome numbers in control conditions (left) or after nocodazole (NOC) treatment (right). Scatter plot with mean (middle bars) and 95% confidence intervals are shown for each group (control: TEC 1–2, n = 52 centrosomes; TEC >2, n = 70 centrosomes; nocodazole treatment: TEC 1–2, n = 55 centrosomes; TEC >2, n = 57 centrosomes). Statistics: Student’s t test; ***, P < 0.001. (D) Diagram of setup that allowed centrosome or control-ablated cells to be relocated. (E) Radial integrated protein intensities of γ-tubulin between various groups. Scatter plot with mean (middle bars) and 95% confidence intervals are shown (one to two centrosomes, n = 26 centrosomes; noncentrosome ablation, n = 43 centrosomes; greater than two centrosomes, n = 29 centrosomes; after supernumerary centrosome ablation, n = 20 centrosomes). Statistics: Student’s t test; ***, P < 0.0001. (F) G1/S-arrested TECs with one to two (left) or greater than two (right) centrosomes in control conditions (top) or with BI2536 treatment and stained for γ-tubulin, pericentrin, and DNA (DRAQ7). Boxes show areas of greater magnification. Numbered yellow lines, line scans of γ-tubulin intensity graphed on the right. Bars: (main images) 5 µm; (insets) 2.5 µm. (G) Radial integrated densities of γ-tubulin in TECs of the indicated centrosome numbers in control conditions (left) or after Bl2536 treatment (right). Scatter plot with mean (middle bars) and 95% confidence intervals are shown (control: TEC 1–2, n = 28 centrosomes; TEC >2, n = 47 centrosomes; Bl2536 treatment: TEC 1–2, n = 63 centrosomes; TEC >2, n = 47 centrosomes). Statistics: Student’s t test; ***, P < 0.001. (H) G1/S-arrested TECs containing greater than two centrosomes in control conditions (left) or expressing Plk1::EGFP WT (right) and stained for (γ-tubulin) and DNA (DRAQ7). Numbered yellow lines, line scans of γ-tubulin intensity graphed to the right. Bars, 10 µm. (I) Mean γ-tubulin radial integrated protein density between the indicated groups normalized to controls. (TEC 1–2, n = 27 centrosomes; TEC >2, n = 27 centrosomes). Statistics: Student’s t test; error bars show means ± SEM; *, P < 0.05. (J) Mean centrosome perimeter between indicated groups. (TEC 1–2, n = 12 cells; TEC >2, n = 15 cells). Statistics: Student’s t test; error bars show means ± SEM; **, P < 0.01. (K) G1/S-arrested TECs containing greater than two centrosomes in control conditions (left) or expressing Plk1::EGFP USN (destruction box mutant, right) and stained for (γ-tubulin) and DNA (DRAQ7). Numbered yellow lines, line scans of γ-tubulin intensity graphed to the right. White asterisk denotes cluster of excess centrosomes. Bars, 10 µm. (L) Mean γ-tubulin radial integrated protein density between indicated groups normalized to controls. (TEC 1–2, n = 29 centrosomes; TEC >2, n = 25 centrosomes). Statistics: Student’s t test; error bars show means ± SEM; *, P < 0.05. (M) Mean centrosome perimeter between indicated groups. (TEC 1–2, n = 8 cells; TEC >2, n = 10 cells). Student’s t test; error bars show means ± SEM; **, P < 0.01. (N, left) Time-lapse fluorescence micrographs of G1/S-arrested TECs with one to two or greater than two centrosomes (cent) and expressing Plk1::EGFP WT (first and third rows), centrin::tdTomato (second row), or Plk1::EGFP USN (fourth row). Colored arrows, centrosomes. (right) Plots of centrosome perimeter over time. Bar, 5 µm. AU, arbitrary unit; Ctrl, control.

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