This time period, 0.1 mg/ml of MTT (3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

This time period, 0.1 mg/ml of MTT (3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in dimethyl sulfoxide was added to 3 wells of each cell type, starting at 0 h, in 24 h intervals. Absorbance was quantified at 540 nm.Soft Agar Colony Formation Title Loaded From File AssayThe soft agar assay was carried out as previously described [7]. Three independent experiments were performed, each one in triplicate.shRNACells were infected with pLKO-based (Open Biosystems) lentiviral vector with or without the human TP53, CBLC or VAV1- shRNA encoding sequences (Table S1). Transfected cells were selected with puromycin.Proteasome InhibitionProteasome inhibition was carried out using 10 mM MG132 (carbobenzoxy-Leu-Leu-leucinal) inhibitor (AGC Scientific, CA, USA). Cells were lysed after 4 hr incubation and subjected to immunoblotting as described above.TUNEL AssayIn Situ Cell Death Detection Kit was purchased (Roche Applied Science, USA) and used according to manufacturer’s instructions.Statistical AnalysisUnpaired Student’s t-test was used to evaluate statistical significance.Results Vav1 is Expressed in the Majority of Human Breast TumorsWe assessed Vav1 expression using a commercial human breast tissue array containing 70 cases of reactive, premalignant and malignant tumors of Eas green bars represent genes whose transcripts were detected at ,103 copies various grades and stages and five normal controls in duplicates. 32 of tumors were estrogen receptor (ER)Vav1 in Breast CancerVav1 in Breast CancerFigure 4. Vav1 as a signal transducer protein in breast cancer cells. (A) MCF-7Vector, MCF-7Vav1, AU565Vector and AU565Vav1 were stimulated with EGF or CSF1, respectively, for various times as indicated. Cell lysates were immunoprecipitated with anti-Vav1 antibody and then immunoblotted with either anti-Vav1 antibody or anti- pTyr antibody (top 2 immunoblots). In addition, total cell lysates were separated on SDS-PAGE and immunoblotted with anti-Vav1, anti-pERK or anti-ERK antibodies (lower 3 immunoblots). (B) Immunofluorescence of 145 MCF-7Vector, 176 MCF7Vav1, 355 AU565Vector and 174 AU565Vav1 with anti-Vav1 antibody. Actin filaments were detected by phalloidin and nuclei were stained with Hoechst. The difference in morphology between MCF-7Vav1, AU565Vav1 and their corresponding control cells were highly significant (two-tailored pValue; 0.0002 and 0.0024 respectively). Representative photographs taken with a Zeiss LSM 710 confocal microscope and analyzed by the ZEN 2010 program are shown. (C) MCF-7Vector, MCF-7Vav1, AU565Vector and AU565Vav1 were transiently transfected with Flag-Rac. 48 hours later, cell lysates were incubated with GST AK bacterial fusion proteins immobilized on glutathione sepharose beads. Bound proteins (+) and unbound proteins (2) were separated on SDS AGE and immunoblotted with anti-Flag mAbs. Numbers indicate mean (+/2 S.E.) relative binding from three different experiments. Unpaired Student’s t-test was used. (*) indicates p,0.05 value. doi:10.1371/journal.pone.0054321.gVav1 as a Signal Transducer in Breast Cancer Cell LinesVav1 is found to be expressed in a large proportion of human breast tumors illustrating its potential huge importance in breast cancer biology. Accordingly, we find mRNA expression of Vav1 is many breast cancer cell lines (Fig. 2A, Table S4); surprisingly we find little or no Vav1 protein mainly due to degradation by Cbl-c. This suggests the existence of complex mechanisms or regulation of Vav1 expression in breast tumors in vivo. To overcome this hurdle for studying the functional role of Va.This time period, 0.1 mg/ml of MTT (3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in dimethyl sulfoxide was added to 3 wells of each cell type, starting at 0 h, in 24 h intervals. Absorbance was quantified at 540 nm.Soft Agar Colony Formation AssayThe soft agar assay was carried out as previously described [7]. Three independent experiments were performed, each one in triplicate.shRNACells were infected with pLKO-based (Open Biosystems) lentiviral vector with or without the human TP53, CBLC or VAV1- shRNA encoding sequences (Table S1). Transfected cells were selected with puromycin.Proteasome InhibitionProteasome inhibition was carried out using 10 mM MG132 (carbobenzoxy-Leu-Leu-leucinal) inhibitor (AGC Scientific, CA, USA). Cells were lysed after 4 hr incubation and subjected to immunoblotting as described above.TUNEL AssayIn Situ Cell Death Detection Kit was purchased (Roche Applied Science, USA) and used according to manufacturer’s instructions.Statistical AnalysisUnpaired Student’s t-test was used to evaluate statistical significance.Results Vav1 is Expressed in the Majority of Human Breast TumorsWe assessed Vav1 expression using a commercial human breast tissue array containing 70 cases of reactive, premalignant and malignant tumors of various grades and stages and five normal controls in duplicates. 32 of tumors were estrogen receptor (ER)Vav1 in Breast CancerVav1 in Breast CancerFigure 4. Vav1 as a signal transducer protein in breast cancer cells. (A) MCF-7Vector, MCF-7Vav1, AU565Vector and AU565Vav1 were stimulated with EGF or CSF1, respectively, for various times as indicated. Cell lysates were immunoprecipitated with anti-Vav1 antibody and then immunoblotted with either anti-Vav1 antibody or anti- pTyr antibody (top 2 immunoblots). In addition, total cell lysates were separated on SDS-PAGE and immunoblotted with anti-Vav1, anti-pERK or anti-ERK antibodies (lower 3 immunoblots). (B) Immunofluorescence of 145 MCF-7Vector, 176 MCF7Vav1, 355 AU565Vector and 174 AU565Vav1 with anti-Vav1 antibody. Actin filaments were detected by phalloidin and nuclei were stained with Hoechst. The difference in morphology between MCF-7Vav1, AU565Vav1 and their corresponding control cells were highly significant (two-tailored pValue; 0.0002 and 0.0024 respectively). Representative photographs taken with a Zeiss LSM 710 confocal microscope and analyzed by the ZEN 2010 program are shown. (C) MCF-7Vector, MCF-7Vav1, AU565Vector and AU565Vav1 were transiently transfected with Flag-Rac. 48 hours later, cell lysates were incubated with GST AK bacterial fusion proteins immobilized on glutathione sepharose beads. Bound proteins (+) and unbound proteins (2) were separated on SDS AGE and immunoblotted with anti-Flag mAbs. Numbers indicate mean (+/2 S.E.) relative binding from three different experiments. Unpaired Student’s t-test was used. (*) indicates p,0.05 value. doi:10.1371/journal.pone.0054321.gVav1 as a Signal Transducer in Breast Cancer Cell LinesVav1 is found to be expressed in a large proportion of human breast tumors illustrating its potential huge importance in breast cancer biology. Accordingly, we find mRNA expression of Vav1 is many breast cancer cell lines (Fig. 2A, Table S4); surprisingly we find little or no Vav1 protein mainly due to degradation by Cbl-c. This suggests the existence of complex mechanisms or regulation of Vav1 expression in breast tumors in vivo. To overcome this hurdle for studying the functional role of Va.