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. Author manuscript; available in PMC: 2008 Oct 2.
Published in final edited form as: Cell Cycle. 2008 May 14;7(14):2225–2233. doi: 10.4161/cc.7.14.6303

Figure 2. Deficiency of MDC1 or BRIT1 led to centrosome amplification.

Figure 2

(A) U2OS cells grown on cover slips were mock-transfected or transfected with luciferase or MDC1 siRNA. At 48 h after transfection, cells were fixed with methanol/acetone (1:1) and costained with antibodies against γ-tubulin and MDC1 followed by Alexa Fluor dye-conjugated secondary antibodies. Nuclei were visualized with DAPI staining. Scale bar, 20 µm. (B) Bar diagram showing that 20% of MDC1-depleted cells contained more than two centrosomes, compared with 3% of luciferase siRNA-treated cells. (C) U2OS cells were treated as described above except that wild-type pEGFP-C2-MDC1 and mutant pEGFP-C2-ΔFHA MDC1 were cotransfected with siRNA to rescue the MDC1-deficient phenotype. Scale bar, 20 µm (D) U2OS cells were grown on cover slips and mock-transfected or transfected with luciferase or MDC1 siRNAs. At 48 h after transfection, cells were treated with 2 mM hydroxyurea for an additional 48 h. Cells were then fixed and costained with antibodies against γ-tubulin and MDC1 followed by Alexa Fluor dye-conjugated secondary antibodies. The percentage of cells containing more than two centrosomes with and without hydroxyurea treatment was determined. (E) U2OS cells grown as described above except that cells were treated with 10 Gy of γ-radiation instead of hydroxyurea. The percentage of cells containing more than two centrosomes after radiation treatment was determined. (F) U2OS cells grown on cover slips were mock-transfected or transfected with luciferase or BRIT1 siRNA. At 48 h after transfection, cells were fixed with methanol/acetone (1:1) and were costained with antibodies against γ-tubulin and BRIT1 followed by Alexa Fluor dye-conjugated secondary antibodies. Nuclei were visualized with DAPI staining. Arrow indicates centrosome amplification with defective cytokinesis.

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