Journal: PLoS ONE

Article Title: BDNF Is Associated with SFRP1 Expression in Luminal and Basal-Like Breast Cancer Cell Lines and Primary Breast Cancer Tissues: A Novel Role in Tumor Suppression?

doi: 10.1371/journal.pone.0102558

Figure Lengend Snippet: Stable clones have been generated with a full-length SFRP1 cDNA or with empty pEF6/V5 vector control. (A) Semi-quantitative real-time PCR for SFRP1 re-expression was performed after transfection in BT20 and (B) SKBR3 cells. SFRP1 mRNA was only detectable in the SFRP1 clones. (C) Western blot analysis was performed on lysates of three BT20 and (D) SKBR3 mock and of three SFRP1 clones. SFRP1 protein expression increased remarkably after transfection with a SFRP1 expression vector compared to the corresponding mock vector. β-actin was used as a loading control.

Article Snippet: The human breast cancer cell lines BT20 and SKBR3 were obtained from the ATCC (Rockville, MD, USA) and cultured under recommended conditions.

Techniques: Clone Assay, Generated, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Transfection, Western Blot

Journal: PLoS ONE

Article Title: BDNF Is Associated with SFRP1 Expression in Luminal and Basal-Like Breast Cancer Cell Lines and Primary Breast Cancer Tissues: A Novel Role in Tumor Suppression?

doi: 10.1371/journal.pone.0102558

Figure Lengend Snippet: Genes represented have a p value of 0.05 or less and are regulated at least ±2-fold. (A) 87 differentially expressed genes were found by comparing BT20/SFRP1 and BT20/mock cells. (B) Comparison of SKBR3/SFRP1 and SKBR3/mock cells revealed 104 differentially expressed genes. (C) By applying class comparison between SFRP1 clones (BT20 and SKBR3) and mock clones (BT20 and SKBR3) 40 differentially expressed genes were discovered. (D) Validation of SFRP1 target genes in SKBR3 and BT20 model system. Semi-quantitative real-time PCR was performed for each target gene in the particular in vitro model. BDNF mRNA levels increased in SKBR3/SFRP1 clones compared to the mock controls (p<0.05). In contrast LY96 mRNA was up-regulated in SKBR3 mock clones (p<0.05). BT20 cells showed an increase of BDNF mRNA levels after SFRP1 re-expression (p<0.05).

Article Snippet: The human breast cancer cell lines BT20 and SKBR3 were obtained from the ATCC (Rockville, MD, USA) and cultured under recommended conditions.

Techniques: Clone Assay, Real-time Polymerase Chain Reaction, In Vitro, Expressing

Journal: PLoS ONE

Article Title: BDNF Is Associated with SFRP1 Expression in Luminal and Basal-Like Breast Cancer Cell Lines and Primary Breast Cancer Tissues: A Novel Role in Tumor Suppression?

doi: 10.1371/journal.pone.0102558

Figure Lengend Snippet: Selected GO categories of the microarray analysis of the basal-like BT20 tumor model.

Article Snippet: The human breast cancer cell lines BT20 and SKBR3 were obtained from the ATCC (Rockville, MD, USA) and cultured under recommended conditions.

Techniques: Microarray

Journal: PLoS ONE

Article Title: BDNF Is Associated with SFRP1 Expression in Luminal and Basal-Like Breast Cancer Cell Lines and Primary Breast Cancer Tissues: A Novel Role in Tumor Suppression?

doi: 10.1371/journal.pone.0102558

Figure Lengend Snippet: (A) Stable cell clones with a full-length cDNA of BDNF show abundant re-expression of BDNF mRNA while empty pT-Rex-DEST30 vector controls completely lack BDNF mRNA. (B) In concordance, mock clones are negative for BDNF protein whereas the 25 kDa BDNF protein is strongly expressed in stable BDNF clones. (C) XTT assay was performed at four subsequent time points. The baseline level at 24 h for each clone was set to 1. A slight decrease in cell proliferation (58.4%) was observed in the stable BDNF clones. (D) Proliferation is significantly (p<0.001) reduced in BT20 breast cancer cells re-expressing BDNF.

Article Snippet: The human breast cancer cell lines BT20 and SKBR3 were obtained from the ATCC (Rockville, MD, USA) and cultured under recommended conditions.

Techniques: Stable Transfection, Clone Assay, Expressing, Plasmid Preparation, XTT Assay

Journal: ISRN Biochemistry

Article Title: Withaferin A Induces Proteasome-Dependent Degradation of Breast Cancer Susceptibility Gene 1 and Heat Shock Factor 1 Proteins in Breast Cancer Cells

doi: 10.5402/2012/707586

Figure Lengend Snippet: A and B: Effects of WA on MDA-MB-231 (a) and BT20 (b) cell apoptosis as measured by Annexin V-FITC-propidium iodide flow cytometry. Cells were treated with indicated concentrations of WA for 24 h. Results were presented as mean ( n = 3) SD. ∗, P < 0.05, significantly different from control by one-way ANOVA. (C) Western analysis of the expression of PARP and caspase-3 in MDA-MB-231 and BT20 cells. Cells were treated with indicated concentrations of WA for 24 h. Western for β -actin was conducted to confirm equal loading of proteins.

Article Snippet: Triple negative MDA-MB-231 and BT20 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM containing 5% fetal bovine serum (FBS), 100 units/Ml penicillin, 100 μ g/mL streptomycin, and 2 mM glutamine.

Techniques: Flow Cytometry, Western Blot, Expressing

Journal: ISRN Biochemistry

Article Title: Withaferin A Induces Proteasome-Dependent Degradation of Breast Cancer Susceptibility Gene 1 and Heat Shock Factor 1 Proteins in Breast Cancer Cells

doi: 10.5402/2012/707586

Figure Lengend Snippet: A. Western analysis of the expression of HSF1 and BRCA1 proteins in MDA-MB-231 and BT20 cells. Cells were treated with indicated concentrations of WA for 24 h.

Article Snippet: Triple negative MDA-MB-231 and BT20 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM containing 5% fetal bovine serum (FBS), 100 units/Ml penicillin, 100 μ g/mL streptomycin, and 2 mM glutamine.

Techniques: Western Blot, Expressing

Journal: Cancer research

Article Title: Activation of Robo1 signaling of breast cancer cells by Slit2 from stromal fibroblast restrains tumorigenesis via blocking PI3K/Akt/?-catenin pathway

doi: 10.1158/0008-5472.CAN-12-0877

Figure Lengend Snippet: A, B. Soft agar colony formation assays using MDA-MB-231 (A) and SKBR3 cells (B) co-cultured with primary carcinoma-associated fibroblasts (CAF) or normal breast-associated fibroblasts (NAF). C, D. Soft agar colony formation assays using breast cancer cell lines other than MDA-MB-231 and SKBR3 co-cultured with immortalized human CAF (199Ct). Colony formation in MDA-MB-361, Hs578T and MDA-MB-157 cells was inhibited (C), but increased in HCC1937, BT20 and MDA-MB-468 cells (D) when co-cultured with 199Ct. E, F. Soft agar colony formation assays using MDA-MB231 (E) and SKBR3 cells (F) co-cultured with two other human fibroblast cell lines, WI38 and Hs68. All data points were performed in triplicates and all experiments were performed at least three times with similar results. Data show means ± standard deviation. **, p < 0.01

Article Snippet: Cell lines and primary fibroblast isolation Human breast cancer cell lines BT20, HCC1937, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, Hs578T, and SKBR3 were obtained from the American Type Culture Collection and maintained in DMEM or DMEM/F12 supplemented with 10% FBS and antibiotics.

Techniques: Cell Culture, Standard Deviation

Journal: Cancer research

Article Title: Activation of Robo1 signaling of breast cancer cells by Slit2 from stromal fibroblast restrains tumorigenesis via blocking PI3K/Akt/?-catenin pathway

doi: 10.1158/0008-5472.CAN-12-0877

Figure Lengend Snippet: A. Immunoblotting analysis of Robo1 expression in MDA-MB-231 cells infected by two independent lentiviruses, #180 and #248, carrying shRobo1. Cells infected with lentiviral sh-luciferease (shCtrl) or uninfected (Mock) served as controls. α-tubulin was used as a loading control. B. Soft-agar colony formation assays using Mock, shCtrl and shRobo1 MDA-MB-231 cells co-cultured with 199Ct. C. Tumor growth assay in NOD/SCID mice. shCtrl or shRobo1 MDA-MB-231 cells were injected into mammary fat-pad with or without 199Ct and the tumor volumes were measured every 4–5 days. D. Immunoblotting analysis of Robo1 expression in MDA-MB-157 cells infected by shRobo1 #180. Cells infected with shCtrl served as the control. α-tubulin was used as a loading control. E. Soft-agar colony formation assays using shCtrl and shRobo1 MDA-MB-157 cells co-cultured with 199Ct. F. Immunoblotting analysis of ectopic expression of Robo1 in BT20 and HCC1937 cells. Cells were infected either with lentiviruses carrying Robo1 cDNA (Robo1) or empty vector (LentiV). β-actin was used as a loading control. G. Soft agar colony formation assay for LentiV and Robo1 overexpressing BT20 and HCC1937 cells co-cultured with or without 199Ct. H. Tumor growth assay in NOD/SCID mice. HCC1937 cells expressing Robo1 or control (LentiV) were injected into mammary fat-pad with or without 199Ct and the tumor volumes were measured every 4–5 days. All data points were performed in triplicates and all in vitro experiments were performed at least three times. Data show means ± standard deviation. For tumor growth assay in NOD/SCID mice, six mice per group were used. *, p < 0.05; **, p < 0.01.

Article Snippet: Cell lines and primary fibroblast isolation Human breast cancer cell lines BT20, HCC1937, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, Hs578T, and SKBR3 were obtained from the American Type Culture Collection and maintained in DMEM or DMEM/F12 supplemented with 10% FBS and antibiotics.

Techniques: Western Blot, Expressing, Infection, Cell Culture, Growth Assay, Injection, Plasmid Preparation, Soft Agar Assay, In Vitro, Standard Deviation

Journal: Cancer research

Article Title: Activation of Robo1 signaling of breast cancer cells by Slit2 from stromal fibroblast restrains tumorigenesis via blocking PI3K/Akt/?-catenin pathway

doi: 10.1158/0008-5472.CAN-12-0877

Figure Lengend Snippet: A. Slit2 mRNA expression in breast cancer cell lines (MDA-MB-231, MDA-MB-361, SKBR3 and BT20), fibroblast cell lines (199Ct, WI38 and HS68) and primary fibroblasts (221C, 222N, 288N and 428N) were examined by qRT-PCR. B. IHC staining with antibody against Slit2 in breast cancer specimen. Low magnification (left panel) scale bar: 25 µm. High magnification (right panel) scale bar: 50 µm. Arrows indicate stromal fibroblasts. Arrow heads indicate cancer cells. C. Soft-agar colony formation assay for MDA-MB-231 cells treated with conditioned medium (199Ct cm) collected from 199Ct culture. D. Immunoblotting analysis of Slit2 proteins in harvested conditioned medium (cm-Slit2) from shCtrl or shSlit2 199Ct. β-actin was used as a loading control. E. Soft agar colony formation assay for MDA-MB-231 cells co-cultured with shCtrl or shSlit2 199Ct. F. Soft agar colony formation of shCtrl and shRobo1 MDA-MB-231 cells treated with PBS or 50 ng/ml of recombinant Slit2 (rSlit2) protein. All data points were performed in triplicates and all experiments were performed at least three times. Data show means ± standard deviation. *, p < 0.05; **, p < 0.01.

Article Snippet: Cell lines and primary fibroblast isolation Human breast cancer cell lines BT20, HCC1937, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, Hs578T, and SKBR3 were obtained from the American Type Culture Collection and maintained in DMEM or DMEM/F12 supplemented with 10% FBS and antibiotics.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Soft Agar Assay, Western Blot, Cell Culture, Recombinant, Standard Deviation

Journal: Cancer research

Article Title: Activation of Robo1 signaling of breast cancer cells by Slit2 from stromal fibroblast restrains tumorigenesis via blocking PI3K/Akt/?-catenin pathway

doi: 10.1158/0008-5472.CAN-12-0877

Figure Lengend Snippet: A. Robo1 overexpressing BT20 cells were treated with rSlit2 (200 ng/ml) for 10, 20 and 30 min. Co-immunoprecipitation (Co-IP) of Robo1 and p85 was detected using immunoblotting analysis in response to the rSlit2 treatment. Normal IgG (IgG) was used as a negative Co-IP control. β-actin served as a loading control. B. Immunoblotting analysis of phospho-Akt in shCtrl and shRobo1 MDA-MB-231 cells treated with rSlit2 (200 ng/ml). Total Akt was used as a quantification control. β-actin served as a loading control. Relative expression (RE) in phospho-Akt to total Akt protein levels is indicated. C. Immunoblotting assay of β-catenin in cytosolic and nuclear fractions of the shCtrl and shRobo1 MDA-MB-231 cells co-cultured with or without 199Ct. α-tubulin and histone deacetylase (HDAC) were used as cytosolic and nuclear markers, respectively. D. Immunofluorescence staining with antibody against β-catenin for shCtrl and shRobo1 MDA-MB-231 cells after treated with rSlit2 (200 ng/ml). Scale bar: 20µm. Arrows indicate nuclei. E. Immunoblotting analysis of shCtrl and shRobo1 MDA-MB-231 cells treated with rSlit2 using antibodies against cyclin D1 and c-myc. α-tubulin as a loading control. RE in cyclin D1 and c-myc to α-tubulin protein levels is indicated. F. Diagram summarized the pathway of how Slit2/Robo1 signal is transmitted from stromal fibroblasts to breast cancer cells.

Article Snippet: Cell lines and primary fibroblast isolation Human breast cancer cell lines BT20, HCC1937, MDA-MB-157, MDA-MB-231, MDA-MB-361, MDA-MB-468, Hs578T, and SKBR3 were obtained from the American Type Culture Collection and maintained in DMEM or DMEM/F12 supplemented with 10% FBS and antibiotics.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Cell Culture, Histone Deacetylase Assay, Immunofluorescence, Staining