Journal: Neoplasia (New York, N.Y.)

Article Title: CAND1 Promotes PLK4-Mediated Centriole Overduplication and Is Frequently Disrupted in Prostate Cancer 1

doi:

Figure Lengend Snippet: CAND1 stabilizes PLK4 and synergizes with PLK4 to induce centriole overduplication. (A) Immunoblot analysis of U-2 OS/centrin-GFP cells for CUL1 and CUL1 deletion mutants. Immunoblot for actin is shown for protein loading. (B) Quantification of the percentage of U-2 OS/centrin-GFP cells with centriole overduplication (more than four per cell and at least one maternal centriole with more than one 1 daughter) following transient transfection with empty vector (control), CUL1, or CUL1 mutants. Mean and standard errors of three independent experiments are shown. Asterisks indicate statistically significant differences (P < .05, Student's t test for independent samples, two-tailed). (C) Cell cycle distribution after transient transfection of U-2 OS/centrin-GFP cells as assessed by flow cytometry. (D, E) Immunoblot analysis of U-2 OS/centrin-GFP cells for PLK4 (anti-myc) or CUL1 mutants (anti-HA) 48 hours after transfection with PLK4-myc, CUL1-ΔN53-HA or CUL1-CΔ22-HA transfection (0-hour CHX), or after 6-hour CHX block. GAPDH is shown to demonstrate protein loading. (F) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells following transfection with either empty vector (control) or PLK4. Note the concurrent formation of multiple daughter centrioles at single maternal centrioles (“centriole flower”), which is highly characteristic for PLK4 overexpression (right panel). Scale bar, 5 µm. (G) Quantification of the percentage of U-2 OS/centrin-GFP cells with centriole overduplication after transfection with PLK4 alone (open bars, µg of PLK4 plasmid DNA transfected as indicated) or in combination with CAND1 (black bars). Mean and standard errors of three independent experiments are shown. Asterisks indicate statistically significant differences (P < .005, Student's t test for independent samples, two-tailed). (H) Immunoblot analysis of U-2 OS/centrin-GFP cells for CAND1 and PLK4 (anti-myc tag), CUL1 or actin after transient transfection of cells with empty myc tag vector (control), CAND1-myc, and/or PLK4-myc and treatment of cells with CHX (60 µg/ml) for the indicated time intervals. Note the increased protein expression of PLK4 in cells cotransfected with CAND1 after 6-hour CHX in comparison to the decreased protein expression when PLK4 is expressed individually.

Article Snippet: Cell Culture and Transfections U-2 OS/centrin-green fluorescent protein (GFP) cells (centrin-GFP construct kindly provided by Michel Bornens, Institut Curie, Paris, France) [ 22 ] were cultured and transiently transfected as previously described [ 12 ].

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Two Tailed Test, Flow Cytometry, Blocking Assay, Fluorescence, Over Expression, Expressing, Comparison