Journal: BMC Cancer

Article Title: Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

doi: 10.1186/1471-2407-11-69

Figure Lengend Snippet: Time course of HER ligand secretion in HME cell lines expressing different levels of HER receptors after addition of 12 ng/ml EGF to the culture medium . Conditioned medium (CM) was collected at 0, 0.5, 2, 8 and 24 h after EGF addition, and ELISA microarray analysis was used to determine the concentrations of A) EGF, B) AREG, C) HB-EGF and D) TGF-α in CM samples from the parental HER1 cell line (diamonds), HER2 (squares, cell line engineered to overexpress HER2 in addition to the endogenous expression levels of HER1 receptor) and HER3 (triangles, a cell line engineered to overexpress HER3 and has endogenous expression levels of HER1). Except at t = 24 h, EGF concentrations were over the detection limit. Therefore, EGF concentrations at early time points are omitted. Data points and crossbars represent the mean and standard deviation of five replicates, each from a separate culture dish. The symbols denote statistically significant differences between the parental HER1 and HER2 (*) or HER3 (#) by both analysis of variance and Fisher's protected least significant difference analysis.

Article Snippet: Spot fluorescent data were then processed and analyzed using Protein Microarray Analysis Tool (ProMAT) and ProMAT Calibrator [ - ], both of which are open-source, freeware programs (available at http://www.pnl.gov/statistics/ProMAT/ ) that we developed specifically for processing ELISA microarray data.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Microarray, Standard Deviation

Journal: BMC Cancer

Article Title: Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

doi: 10.1186/1471-2407-11-69

Figure Lengend Snippet: Time-dependent changes of HER ligand concentrations in cell lysates . HMEC lines expressing different levels of HER receptors were treated with 12 ng/ml EGF for up to 24 h. Cell lysates were collected at t = 0, 0.5, 2, 8 and 24 h, and ELISA microarray analysis was used to determine the concentration of A) AREG and B) TGF-α in cell lysates from the parental HER1 cell line (diamonds), HER2 (squares) and HER3 (triangles). Results are presented as ng ligand per mg cell-lysate protein. Data points and crossbars represent the mean and standard deviation of five replicates, each from a separate culture dish. The symbols denote statistically significant differences between the parental HER1 and HER2 (*) or HER3 (#) by both analysis of variance and Fisher's protected least significant difference analysis.

Article Snippet: Spot fluorescent data were then processed and analyzed using Protein Microarray Analysis Tool (ProMAT) and ProMAT Calibrator [ - ], both of which are open-source, freeware programs (available at http://www.pnl.gov/statistics/ProMAT/ ) that we developed specifically for processing ELISA microarray data.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Microarray, Concentration Assay, Standard Deviation

Journal: BMC Cancer

Article Title: Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

doi: 10.1186/1471-2407-11-69

Figure Lengend Snippet: Effect of MAPK/Erk and PI3K/Akt pathway inhibitors on HER1 ligand secretion . HMEC lines expressing different levels of HER receptors were preincubated with a single inhibitor (20 μg/ml LY 294002, 200 nM wortmannin, 25 µM PD 98059 or 10 µM U0126) for 1 h, followed by treatment with 12 ng/ml EGF for 8 h. DMSO concentration was 0.1% in all experiments except those marked as "ctrl". Conditioned medium (CM) was collected and the concentrations of A) AREG, B) HB-EGF and C) TGF-α were determined by ELISA microarray analysis. Ligand concentrations were normalized with respect to the concentration in the HER1 cells treated only with 12 ng/ml EGF for 8 h. Columns and crossbars represent the mean and standard deviation, respectively, from five replicates, each of which was from a separate culture dish. The standard deviation is shown for every analysis, but in some cases the crossbar may be too small to be visible. The asterisks denote statistically significant differences between inhibitor-treated cells and ctrl cells for the individual cell lines, as determined by both analysis of variance and by Fisher's protected least significant difference.

Article Snippet: Spot fluorescent data were then processed and analyzed using Protein Microarray Analysis Tool (ProMAT) and ProMAT Calibrator [ - ], both of which are open-source, freeware programs (available at http://www.pnl.gov/statistics/ProMAT/ ) that we developed specifically for processing ELISA microarray data.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Microarray, Standard Deviation

Journal: BMC Cancer

Article Title: Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression

doi: 10.1186/1471-2407-11-69

Figure Lengend Snippet: Effects of MAPK/Erk and PI3K/Akt pathway inhibitors on cytokine secretion in HME cell lines that express different levels of HER receptors . All cell lines were preincubated with one inhibitor (20 μg/ml LY 294002, 200 nM wortmannin, 25 µM PD 98059 or 10 µM U0126) for 1 h, followed by treatment with 12 ng/ml EGF for 8 h. DMSO concentration in the culture media was 0.1% in all the experiments except those marked as "ctrl" runs, which lacked any DMSO. CM was collected from the parental HER1, HER2 and HER3 cell lines and concentrations of accumulated A) VEGF and B) PDGF were determined by ELISA microarray analysis. The concentration of each growth factor was normalized with respect to its concentration in the HER1 cells that were treated with 12 ng/ml EGF for 8 h. Error bars represent one standard deviation from the mean of five biological replicates. The asterisks denote statistically significant differences between treated samples and ctrl runs with EGF in each cell line by both analysis of variance and Fisher's protected least significant difference analysis.

Article Snippet: Spot fluorescent data were then processed and analyzed using Protein Microarray Analysis Tool (ProMAT) and ProMAT Calibrator [ - ], both of which are open-source, freeware programs (available at http://www.pnl.gov/statistics/ProMAT/ ) that we developed specifically for processing ELISA microarray data.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Microarray, Standard Deviation