Effects of BRCA1 on p3TP-Lux promoter activity.

<p>(A) Wild type BRCA1 activates the p3TP-Lux promoter. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase plasmids were co-transfected with Flag-Smad3 and HA-BRCA1 plasmids into COS-7 cells, as indicated. A pcDNA3 empty vector plasmid was used to adjust total DNA amounts. At 36 hrs after transfection, luciferase and <i>β</i>-galactosidase assays were performed. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. *, <i>P</i><0.05 versus empty vector only. (B) Wild type BRCA1 enhances TGF-<i>β</i>-mediated p3TP-Lux promoter activity. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase plasmids were co-transfected with 0.25 µg of caT<i>β</i>RII plasmid or 0.1 µg of HA-BRCA1 plasmid into COS-7 cells, as indicated. 24 hrs after transfection, the indicated cells were incubated with 2 ng/ml of TGF-<i>β</i>1 ligand overnight, followed by luciferase and <i>β</i>-galactosidase assays. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. (C) BRCA1 mutants could not activate the p3TP-Lux promoter. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase were co-transfected with 0.1 µg of wild type or various mutant forms of HA-BRCA1 into COS-7 cells. 24 hrs after transfection, the cells were treated with or without 2 ng/ml of TGF-β1 overnight, followed by luciferase and <i>β</i>-galactosidase assays. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. WT: wild type BRCA1; M, P, and Y: BRCA1-M1775R, BRCA1-P1749R, and BRCA1-Y1853x, respectively; N-: BRCA1 (1–683aa); C-: BRCA1 (1301–1863aa).</p

Effects of silencing BRCA1 in MCF-7 cells on Smad3 and Smad4.

<p>(A) si-BRCA1 decreases Smad3-Smad4-mediated transcriptional activation of p3TP-Lux reporter in MCF7 cells. MCF7 cells were transfected with 5 nM of siRNAs in 24 well plate. After 16–24 hours, the cells were transfected with p3TP-Lux, pCMV-β-galactosidase, Smad3, Smad4, and empty vector plasmids as indicated. The cells were maintained in cell culture medium containing 0.1% FBS overnight, followed by luciferase and β-galactosidase assays. The fold increase of luciferase activity was obtained from triplicate of three independent experiments, after being normalized by β-glactosidase activity. The Excel software was used to calculate the standard deviation. * <i>P</i><0.05 as compared to si-GFP treatments+Smad3+Smad4. (B) Depletion of BRCA1 decreases TGF-β-induced Smad3-Smad4 interaction in MCF-7 cells. MCF-7 cells were transfected with 5 nM siGL2 or siBRCA1. After overnight, cells were starved for 3 hours, followed by 1 ng/ml of TGF-β1 treatment for 45 min. Total lysates were subjected to immunoprecipitation with anti-Smad4 antibody and Western blotting with anti-Smad3 antibody as indicated. The membrane was re-probed with anti-Smad4 antibody to monitor the levels of Smad4 protein. The levels of Smad3 protein was detected from 20 µg of total lysatesby using anti-Smad3 antibody. This membrane was reprobed with anti-Actin antibody to monitor equal loading.</p

Effects of H₂O₂ on the TGF-<i>β</i>1-induced association between Smad3 and Smad4.

<p>(A) H₂O₂ increases the interaction between Smad3 and Smad4 in HEK293T cells. HEK293T cells were co-transfected with 0.5 µg of Flag-Smad3, 0.5 µg of HA-Smad4, and 0.15 µg of caT<i>β</i>RII plasmids, as indicated. 24 hours later, cells were treated with 200 µM of H₂O₂ for 2 hours, followed by immunoprecipitation and Western blotting, as indicated. The membrane was re-probed with anti-Flag monoclonal antibody to examine the expression levels of Flag-Smad3 protein. The increases in Flag-Smad3-binding to HA-Smad4 were normalized to Flag-Smad3 protein expression and are represented graphically. The expression levels of HA-Smad4 protein were determined in 10 µg of total cell lysates. This is a representative experiment out of 4 experiments. (B) H₂O₂ increases the interaction between Smad3 and Smad4 in HaCaT cells. HaCaT cells were treated or untreated with 2 ng/ml of TGF-<i>β</i>1 and 100 µM of H₂O₂ for 1 hour as indicated, and subjected to immunoprecipitation with anti-Smad3 polyclonal antibody and Western blotting with anti-Smad4 monoclonal antibody. The membrane was re-probed with anti-Smad3 antibody to examine the levels of Smad3 protein. The increases in Smad3-binding to Smad4 were normalized to Smad3 protein expression and are represented graphically. The expression levels of Smad4 protein were determined in 20 µg of total cell lysates. This is a representative experiment out of 4 experiments. (C) TGF-<i>β</i>1-induced Smad3 and Smad4 interaction is decreased by H₂O₂ in HCC1937 cells. HCC1937 cells were treated or untreated with 2 ng/ml of TGF-<i>β</i>1 and 100 (+), 200 (++), and 300 µM (+++) of H₂O₂ for 1 hour as indicated. Immunoprecipitation was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007091#pone-0007091-g005" target="_blank">Figure 5b</a> (HaCaT cells). The membrane was re-probed with anti-Smad3 antibody to examine the levels of Smad3 protein. The decreases in Smad3-binding to Smad4 were normalized to Smad3 protein expression and are represented graphically. The expression levels of Smad4 protein were determined in 20 µg of total cell lysates. This is a representative experiment out of 4 experiments. (D) Wild type BRCA1 restores the TGF-<i>β</i>1-induced Smad3 and Smad4 interaction against H₂O₂ in HCCBRCA1 cells. The experiment was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007091#pone-0007091-g005" target="_blank">Figure 5c</a> (HCC1937 cells). The increases in Smad3-binding to Smad4 were normalized to Smad3 protein expression and are represented graphically. This is a representative experiment out of 4 experiments.</p

Effects of BRCA1 on Smad3 protein.

<p>(A) Wild type BRCA1 and Smad3 cooperate to increase p3TP-Lux promoter activity. 0.1 µg of p3TP-Lux and 10 ng of pCMV<i>β</i>-galactosidase plasmids were co-transfected with 10 ng of Flag-Smad3, 50 ng of HA-Smad4, and 0.1 µg of wild type HA-BRCA1 plasmids into COS-7 cells, as indicated. 36 hrs after transfection, luciferase and <i>β</i>-galactosidase assays were performed. The luciferase activities were normalized to <i>β</i>-galactosidase activity and the relative luciferase activities (fold increase) were calculated as the ratio of the normalized luciferase activity with effectors to that without effectors. The average±S.D. was calculated from triplicate plates and experiments were repeated three times with similar results. The bars in the graphs represent mean±SD. *, <i>P</i><0.05 versus empty vector only. (B) Wild type BRCA1 increases Smad3 protein abundance. 0.15 µg of Flag-Smad3 plasmid was co-transfected with 0.1 µg, 0.25 µg, and 0.5 µg of wild type HA-BRCA1 or 0.1 µg and 0.25 µg of caT<i>β</i>RII into HEK293 cells. 36 hrs after transfection, 10 µg of total cell lysates was subjected to Western blotting with anti-Flag monoclonal antibody. Anti-CSK polyclonal antibody was used to monitor equal loading. Flag-Smad3 expression levels were normalized to CSK and are represented graphically. This is a representative experiment out of 4 experiments. (C) BRCA1 mutants could not increase Smad3 protein abundance. 0.15 µg of Flag-Smad3 was co-transfected with 0.1 µg of wild type or various mutant forms of HA-BRCA1 into HEK293 cells. 10 µg of total cell lysates was subjected to Western blotting with anti-Flag monoclonal antibody. Anti-CSK polyclonal antibody was used to monitor equal loading. Flag-Smad3 expression levels were normalized to CSK and are represented graphically. This is a representative experiment out of 4 experiments.</p

Effects of H₂O₂ on the trafficking of phospho-Smad3 protein.

<p>(A) H₂O₂ induces the nuclear export of TGF-<i>β</i>1-activated phospho-Smad3 protein (p-Smad3) in HCC1937 cells, but not in HCCBRCA1 or HaCaT cells. Cells were pretreated with 2 ng/ml of TGF-<i>β</i>1 for 1 hour and were then incubated with or without 100 µM of H₂O₂ for an additional 1 hour. Cytosolic and nuclear proteins were subjected to Western blotting with anti-phospho-Smad3 antibody. The membranes were re-probed with anti-GAPDH and anti-Lamin B1 antibodies to monitor fractionation efficiency and equal loading for the cytosolic and nuclear proteins. Increases or decreases in the amount of phospho-Smad3 protein were normalized to the amount of GAPDH and Lamin B1 protein in the cytosol and nucleus, respectively. Cyt.: cytosolic fraction; Nuc.: nuclear fraction. This is a representative experiment out of 4 experiments. (B) Knockdown of endogenous BRCA1 in HCCBRCA1 cells sensitizes phospho-Smad3 protein to H₂O₂ and induces redistribution of the protein into the cytoplasm. HCCBRCA1 cells were transfected with siRNAs against BRCA1 and GL2 (control). 48 hours later, cells were pretreated with 2 ng/ml of TGF-<i>β</i>1 for 1 hour and were then incubated with or without 100 µM of H₂O₂ for an additional 1 hour. Cytosolic and nuclear proteins were subjected to Western blotting with anti-phospho-Smad3 antibody (p-Smad3). The membranes were re-probed with anti-Lamin B1 and anti-GAPDH antibodies, as indicated, to monitor fractionation efficiency and equal loading. The changes in the amount of phospho-Smad3 protein were normalized to the amount of GAPDH and Lamin B1 protein in the cytosol and nucleus, respectively. The right panel shows the knockdown of nuclear BRCA1 protein after siRNA against BRCA1. Cyt.: cytosolic fraction; Nuc.: nuclear fraction. This is a representative experiment out of 4 experiments.</p

Effects of H₂O₂ on the association and colocalization between Smad3 and BRCA1.

<p>(A & B) H₂O₂ increases Smad3 and BRCA1 interaction in HaCaT and HCCBRCA1 cells. Cells were treated with TGF-<i>β</i>1 (2 ng/ml) and H₂O₂ (100 µM) for 1 hour, as indicated. Total cell lysates were subjected to immunoprecipitation with anti-Smad3 polyclonal antibody and Western blotting with anti-BRCA1 polyclonal antibody. The increases in Smad3-binding to BRCA1 were normalized to Smad3 protein expression and are represented graphically. The expression levels of Smad3 and BRCA1 proteins were determined in 20 and 30 µg of total cell lysates, respectively. The shifted bands of BRCA1 after H₂O₂ treatment are shown in HaCaT cells. This is a representative experiment out of 4 experiments. (C) H₂O₂ increases phospho-Smad3 and BRCA1 colocalization in HCCBRCA1 cells. HCCBRCA1 cells were treated with 2 ng/ml of TGF-<i>β</i>1 and 100 µM of H₂O₂ for 1 hour as indicated. Anti-BRCA1 monoclonal antibody (Ab-3) and anti-phospho-Smad3 polyclonal antibody were used to immunostain BRCA1 protein (Red) and phospho-Smad3 protein (Green), respectively. Merge shows the colocalization of BRCA1 and phospho-Smad3 protein. This is a representative experiment out of 4 experiments.</p

BRCA1 and Smad3 association.

<p>(A) HA-BRCA1 interacts with Flag-Smad3 in HEK293T cells. 0.5 µg of wild type HA-BRCA1 and 0.50 µg of Flag-Smad3 plasmids were co-transfected into HEK293T cells, as indicated. 36 hrs later, total cell lysates were subjected to immunoprecipitation and Western blotting with anti-Flag monoclonal antibody and anti-HA-monoclonal antibody, as indicated. 10 µg of total cell lysates was used to examine the expression levels of Flag-Smad3 and HA-BRCA1 protein. The blots are representative experiment out of 3 experiments. (B) TGF-<i>β</i>1 increases the interaction between HA-BRCA1 and Flag-Smad3 in COS-7 cells. 0.50 µg of Flag-Smad3 and 0.50 µg of HA-BRCA1 plasmids were co-transfected into COS-7 cells. 24 hrs later, cells were treated with or without 2 ng/ml of TGF-<i>β</i>1 as indicated. Total cell lysates were immunoprecipitated with anti-Flag monoclonal antibody followed by Western blotting with anti-HA monoclonal antibody. The membrane was re-probed with anti-Flag antibody. The increases in HA-BRCA1 were normalized to Flag-Smad3 and are represented graphically. The expression levels of HA-BRCA1 protein were detected in 10 µg of total cell lysates. This is a representative experiment out of 3 experiments. (C) TGF-<i>β</i>1 increases BRCA1 and Smad3 interaction in MCF-7 cells. Cells were stimulated with 2 ng/ml of TGF-<i>β</i>1 for 1 hour. Total cell lysates were immunoprecipitated by anti-Smad3 polyclonal antibody, followed by Western blotting with anti-BRCA1 antibody. The membrane was re-probed with anti-Smad3 antibody. The increases in BRCA1 were normalized to Smad3 and are represented graphically. The expression levels of BRCA1 protein were determined in 30 µg of total cell lysates. This is a representative experiment out of 4 experiments. (D) Smad3 binding site in BRCA1. 10 µg of the individual GST-BRCA1 protein fragments and HEK293T cell lysates transfected with Flag-Smad3 plasmid were subjected to a GST-pull down assay. To monitor expression levels, 10 µg of individual GST-BRCA1 protein fragments were separated on 15% and 8% SDS-PAGE, followed by Coomassie staining. (E) Interaction of BRCA1 (1–683aa) with Smad3 protein in HEK293T cells. 0.5 µg of HA-<i>BRCA1</i>(1–683aa) and 0.5 µg of <i>Smad3</i> plasmids were co-transfected into HEK293T cells, followed by immunoprecipitation and Western blotting, as indicated. The membrane was re-probed with anti-Flag monoclonal antibody to examine the levels of Smad3 protein expression. The levels of HA-BRCA1(1–683aa) expression were determined in 10 µg of total lysates. (F) BRCA1 binds to the MH2 domain in Smad3. 10 µg of GST and GST-BRCA1 (298–436aa) proteins and HEK293T cell lysates transfected with Flag-Smad3 (1–206aa) or Flag-Smad3 (207–426aa) were subjected to a GST pull-down assay. The expression levels of Flag-Smad3 (1–206aa) and Flag-Smad3 (207–426aa) protein were determined in 10 µg of total cell lysates. This is a representative experiment out of 4 experiments. (G) Flag-Smad3 and HA-BRCA1 (298–436aa) interaction. HA-BRCA1 (298–436aa) and Flag-Smad3 plasmids were co-transfected into HEK293T cells, followed by immunoprecipitation and Western blotting, as indicated. The membrane was re-probed with anti-Flag antibody to monitor Flag-Smad3 expression. 10 µg of total cell lysates was used to examine the expression levels of HA-BRCA1 (298–436aa) protein.</p