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Figure 2 | Breast Cancer Research

Figure 2

From: New role for nuclear hormone receptors and coactivators in regulation of BRCA1-mediated DNA repair in breast cancer cell lines

Figure 2

BRCA1 inhibition decreases DNA damage repair and cell survival in human breast cancer cell lines. (a) Expression of dominant-negative BRCA1 in human breast cancer cell lines. T47D and MDA-MB-468 cells were stably transfected with the BRCA1 carboxyl-terminal truncation mutant (BRCA1delC) or neomycin expression vector (neo). Protein extracts from these clones were subjected to western blotting with anti-BRCA1 antibody. Anti-β-actin antibody was used to determine relative amounts of protein in each lane. Representative blots are shown. (b) BRCA1 inhibition decreases the expression of double-strand break repair proteins in human breast cancer cell lines. T47D or MDA-MB-468 BRCA1delC or neomycin-resistant control (neo) clones were treated with etoposide (etopo) or vehicle prior to western blotting with antibodies indicated at the left. Blots were stripped and incubated with anti-β-actin antibody to determine relative amounts of protein in each lane. Representative blots are shown. (c) T47D and MDA-MB-468 BRCA1delC clones exhibit increased DNA damage when treated with etoposide. BRCA1delC or neomycin-resistant control clones (neo) were pretreated with E2RA alone or in combination (E2/RA) before exposure to etoposide. Vehicle-treated cultures were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. Error bars indicate SEM. (d) T47D and MDA-MB-468 BRCA1delC clones exhibit decreased DNA repair activity. BRCA1delC or neomycin-resistant control clones (neo) were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. Error bars indicate SEM. (e) Increased cell survival in etoposide-treated T47D and MDA-MB-468 BRCA1delC clones. Neomycin control clones were used as controls. Cultures were pretreated with E2 or RA alone or in combination (E2/RA). Vehicle-treated cultures were used as the negative control (con). Cell death was quantified by TdT-mediated dUTP nick end labelling assay. These experiments were repeated three times with similar results. Error bars indicate SEM. (f) No complex formation of BRCA1delC with CBP and estrogen receptor α (ERα) in T47D and MDA-MB-468 cells. Stable clones expressing BRCA1delC were treated with E2 or RA as described in Materials and Methods. Vehicle-treated cells were used as the negative control. The CBP coactivator protein was immunoprecipitated from treated cultures with anti-CBP antibody (IP CBP). Preimmune IgG was used as the negative control for immunoprecipitation. Immunoprecipitates were subjected to western blotting to detect interaction with ERα, retinoic acid receptor α (RARα), and BRCA1 by using the antibodies indicated at the left. Blots were stripped and incubated with anti-CBP antibody to detect relative amounts of immunoprecipitated protein in each lane. In contrast, both wild-type and mutant BRCA1 immunoprecipitated from both cell lines with anti-BRCA1 antibody (lower panel). These experiments were performed three times with similar results; representative blots are shown.

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