Figure 4 (See previous page).

BRCA1 and FancJ cooperatively promote mitomycin C-induced centrosome amplification. (A and B) Co-depletion of BRCA1 and FancJ further attenuates MMC-induced centrosome amplification. U2-OS cells were transfected with either Control siRNA (C), siRNA against BRCA1 (B), FancJ (J), or both BRCA1 and FancJ (B + J) and then split into 2 sets. One set was collected for protein gel blot analysis (A). The second set of cells was treated with 0.5 μM MMC for 72 hours and then fixed in methanol and stained with antibodies against γ-Tubulin. More than 300 cells were counted and the percentage of cells with more than 2 centrosomes was quantified (B). (C and D) Overexpression of BRCA1 or FancJ or both stimulates the MMC-induced centrosome amplification. U2-OS cells were transfected with plasmid expressing either GFP, GFP-BRCA1 (B), FLAG-FancJ (J), or both GFP-BRCA1 and FLAG-FancJ (B + J). 48 hours after transfection, the cells were then pooled and split into 5 sets (Set #1 to #5). Cells of Set #1 were collected for western blot analysis (C). For cells of Sets #2 to #5, they were either left untreated (Set #2, 0 h) or treated with 0.5 μM MMC for the indicated time (Set #3 to #5), and then were fixed in methanol and stained with antibodies against γ-Tubulin. More than 300 cells were counted and the percentage of cells with more than 2 centrosomes was quantitated (D). (E) Inhibition of PLK1 in BRCA1 and FancJ co-depleted cells further attenuates the centrosome amplification defects. U2-OS cells were transfected with either Control siRNA (C) or siRNA against both BRCA1 and FancJ (B + J) and then split into 2 sets. One set of cells was treated with 0.5 μM MMC for 72 hours (-BI). In the second set, cells were first treated with 0.5 μM MMC for 12 hours, followed by the addition of 100 nM BI-2536 (+BI). Sixty hours later, cells were fixed and stained with antibodies against γ-Tubulin. More than 300 cells were counted and the percentage of cells with more than 2 centrosomes was quantified. (F and G) Expression of the constitutive active PLK1 rescues the centrosome amplification defects in BRCA1 and FancJ co-depleted cells. The 3 different U2-OS cell lines expressing different PLK1 variants as in Figure 3 were first transfected with either Control siRNA (C) or siRNA against both BRCA1 and FancJ (B + J). Dox was added to the cells to induce the expression of PLK1 variants. The knockdown efficiency of BRCA1 and FancJ were monitored by protein gel blot (F). In addition, cells were treated with 0.5 μM MMC for 72 hours, then fixed in methanol and stained with antibodies against γ-Tubulin. More than 300 cells were counted and the percentage of cells with more than 2 centrosomes was quantified (G). Immunoblotting antibodies are indicated on the right. All error bars are standard deviation obtained from 3 different experiments. Standard 2-sided t test: *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant.