Figure 1.

Identification of BRCA1 missense mutations that affect regulation of centrosome number. (a) Results of the centrosome amplification assay with BRCA1 substitution variants. Control (lane 1) or BRCA1 3⁰-UTR small-interfering RNA (lanes 2–17) were transfected into Hs578T cells along with GFP-centrin 2 and the indicated BRCA1 mutant expressing plasmid or empty vector. The histogram shows the percentage of Hs578T cells with centrosome amplifications after the substitution of BRCA1 with the indicated BRCA1 variant. Less than 5.5% of the cells with amplified centrosomes was considered normal in this assay, and greater than 10% with amplified centrosomes was considered defective for centrosome regulation. The percentage given for each variant represents the average of three independent repeats of the assay with more than 100 cells counted for each mutant/experiment. The control small-interfering RNA was tested a total of 11 times and the BRCA1si with co-transfected vector plasmid was tested eight times. (b) A histogram showing the number of cells with centrosome amplification (left, same value as in panel a) that also had paired centrioles upon the introduction of the L52F variant as compared with the M18T variant. (c) Immunoblots showing the expression of BRCA1 substitution variants in Hs578T cells. Cells were processed according to the schedule of the centrosome assay and the lysates were analyzed for the expression of BRCA1 (top) or the loading control alpha-tubulin (bottom). In lanes 1, 10 and 17 cells were transfected with the control small-interfering RNA and with empty plasmid vector. In lanes 2, 11 and 18 cells were transfected with BRCA1 3⁰-UTR-specific small-interfering RNA and with empty plasmid vector. In lanes 3–9, 12–16 and 18–21 cells were transfected with BRCA1 3⁰-UTR small-interfering RNA small-interfering RNA along with the indicated substituting BRCA1 variant. Densitometric analysis is included in supplementary Figure S2.