Fig. 1

Identification of BRE as a genetic interactor of BRCA2 using a MSCV-based insertional mutagenesis screen. a Schematic representation of MSCV-mediated insertional mutagenesis in Brca2 conditional mouse ES cells. Brca2^KO/KO cells generated after PGK-CRE-mediated deletion of the conditional allele are not viable. Mutagenesis by MSCV-CRE can generate viable Brca2^KO/KO ES cells. b Southern blot analysis of HAT-resistant ES cell colony that lost conditional Brca2 allele (Brca2^KO/KO; Clone 3d) after MSCV-CRE transduction. Upper band: conditional allele (CKO); lower band: knock-out allele (KO). c Quantification of Bre expression by real-time RT-PCR in Brca2 conditional mutant (Brca2^CKO/KO) and Brca2^KO/KO ES cells with viral insertion at chr: 5qB1 (Brca2^KO/KO;Clone 3d). Data are represented as mean ± s.d. Top panel shows the distance between viral insertion and start of first exon of Bre. d Expression of HA-tagged BRE from MSCV LTR in Brca2^CKO/KO clones. Two independent clones Clone #1 and Clone #2 analyzed by western blot analysis were used further. Left panel shows the scheme of relevant alleles of Brca2^CKO/KO; MSCV-BRE ES cells. e Southern blot analysis of HAT-resistant ES cell colonies after CRE-mediated deletion of conditional allele in Brca2^CKO/KO; MSCV-BRE ES cells to identify Brca2^KO/KO clones (marked with solid stars), upper band: conditional allele (CKO); lower band: knock-out allele (KO). Schematic diagram of CRE-induced loss of conditional Brca2 allele in Brca2^CKO/KO; MSCV-BRE cells is shown at the top. f Upper panel shows western blot for BRCA2 knockdown by two different shRNAs (#1 and #2) and a non-specific (NS) control and HA-BRE expression in MCF7 cells that were stably expressing either empty vector (MCF7Neo) or vector expressing HA-BRE (MCF7BRE). GAPDH was used as a loading control. Growth of MCF7 cells after BRCA2 knockdown in the presence or absence of HA-BRE expression represented in the lower panel. Fold growth was calculated by dividing cell counts on particular day with cell count on day 1. P values are shown in Supplementary Table 2. P values were calculated using paired two-tailed t-test