Journal: Breast Cancer Research : BCR

Article Title: Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells

doi: 10.1186/bcr2895

Figure Lengend Snippet: Primer and siRNA sequences used in this study

Article Snippet: NG2 siRNA (sc-40771) from Santa Cruz Biotechnology, Inc. was used for inhibition of CSPG4.

Techniques: Sequencing

Journal: Breast Cancer Research : BCR

Article Title: Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells

doi: 10.1186/bcr2895

Figure Lengend Snippet: Inhibition of CHST11 expression and P-selectin binding to MDA-MB-231 cells by CHST11 siRNA . MDA-MB-231 cells were treated with three different siRNAs for the CHST11 gene. RNA was harvested after 48 hours and gene expression was assayed at the mRNA level (A) . GAPDH was used as the house keeping gene to normalize mRNA-based expression data using the delta delta CT method. CHST11 mRNA levels are shown relative to mRNA level in cells treated with transfection agent only (vehicle). Data were log transformed and subjected to one way ANOVA with post-hocTukey's analysis. B) Binding of anti-CS-A (2H6 mAb) (top) and P-selectin (bottom) was tested at Day 6 post siRNA transfection. Binding of secondary antibodies only serves as control. Binding of anti-CS-A 2H6 mAb and recombinant human P-selectin to vehicle-treated and siRNA-treated MDA-MB-231 cells with the three siRNAs is shown. Mean fluorescent intensities of three independent experiments were log transformed and analyzed by ANOVA and post-hoc comparison. Treatment with CHST11 siRNA #31 significantly reduced mean fluorescent intensities for anti-CS-A 2H6 mAb ( P ≤ 0.015) and P-selectin ( P ≤ 0.001) binding, as compared with vehicle-treated cells.

Article Snippet: NG2 siRNA (sc-40771) from Santa Cruz Biotechnology, Inc. was used for inhibition of CSPG4.

Techniques: Inhibition, Expressing, Binding Assay, Gene Expression, Transfection, Transformation Assay, Control, Recombinant, Comparison

Journal: Breast Cancer Research : BCR

Article Title: Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells

doi: 10.1186/bcr2895

Figure Lengend Snippet: The expression of CSPG4 in aggressive breast cancer cell lines contributes to P-selectin binding . A) CSPG4 mRNA was measured by qRT-PCR and normalized to 18S values and log transformed. Means and standard deviations are shown. Comparisons were made by ANOVA and post-hoc analysis. B) Cell surface expression of CSPG4 was examined in the indicated breast cancer cell lines by flow cytometry using anti-CSPG4 225.28 mAb (10 μg/ml). Open histogram shows binding of the secondary antibody only, while filled histogram shows anti-CSPG4 225.28 mAb binding. One representative experiment out of three is shown. C) Expression of CSPG4 was inhibited by transient transfection of MDA-MB-231 cells with CSPG4 siRNA that led to a decrease in the binding of P-selectin (D) and anti-CS-A (E) to transfected cells In C, D and E the filled histograms show the binding of secondary antibodies only (control), the open histograms with solid lines show binding to vehicle-treated cells while the open histograms with dotted lines show binding to the siRNA-transfected cells.

Article Snippet: NG2 siRNA (sc-40771) from Santa Cruz Biotechnology, Inc. was used for inhibition of CSPG4.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Transformation Assay, Flow Cytometry, Transfection, Control