Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: 4T1-Her2 cells were seeded in 96 well plates at increasing numbers and the luciferase level was determined after 2 hours. Both cell number and luciferase quantity were converted into percentage by dividing the individual figure with the maximal cell number seeded (100,000) or the luciferase reading from the well with the maximal cell number. The correlation coefficient (R) is indicated in the figure.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase

Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: Initially 10,000 4T1-Her2 cells were seeded. The supernatants were collected at the indicated times for measurement of luciferase.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase

Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: A . Cell-associated and supernatant luciferase activity during cTCR-splenocyte mediated cytolysis of 4T1-Her2 cells. cTCR-splenocytes were added to 4T1-Her2 cells at a ratio of 10∶1. Supernatants and cells were harvested at the indicated time for quantification of luciferase activity. B . 4T1-Her2 monolayer without cTCR-transduced splenocytes. C . Seventy-two h after cTCR-tranduced splenocytes were added to 4T1-Her2 monolayer.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: A Simple and Sensitive Method for Measuring Tumor-Specific T Cell Cytotoxicity

doi: 10.1371/journal.pone.0011867

Figure Lengend Snippet: Mock-transduced ( A ) or cTCR-transduced ( B ) splenocytes were added to 4T1-Her2 cells at the indicated ratio. Cells were harvested 72 later for quantification of luciferase activity. C . Using the formula: % specific luc reduction = (% luc reduction from engrafted T-cell)/(% luc reduction from control T-cell)×100, the data from A and B were converted into percentage of specific luciferase release and plotted.

Article Snippet: The sorted GFP positive cells were then seeded as single cells in 96-well plate by limiting dilution and screened for colonies expressing HER2 by staining them with PE conjugated -anti-human HER2 antibody (BioLegend, San Diego, CA).

Techniques: Luciferase, Activity Assay, Control

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Apoptotic effects of EVOO phenolics in MCF-7 breast cancer cells engineered to overexpress HER2 . Quantification of apoptosis-related cell death in MCF-7/HER2 and MCF-7/pBABE matched control cells treated with increasing concentrations of EVOO phenolics was determined by Cell Death ELISA as described in "Materials and methods". The enrichment of histone-DNA fragments in EVOO polyphenols-treated cells was expressed as fold-increase in absorbance by comparing with control (vehicle-treated) cells using the following formula: [A 405 – A 490 ] TREATED /[A 405 -A 490 ] UNTREATED . Data are the mean ( columns ) and 95% confidence intervals ( bars ) of three independent experiments performed in duplicate. One-factor ANOVA was used to analyze differences in the percentage of apoptosis between HER2-negative MCF-7/pBABE cells and HER2-positive MCF-7/HER2 cells. Statistically significant differences are shown (one-factor ANOVA analysis). All statistical tests were two-sided.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Effects of EVOO phenolics on HER2-enhanced breast cancer cell proliferation . MCF-7/HER2 cells and MCF-7/pBABE matched control cells were incubated with 100 μM of EVOO single phenols ( left ), fractions containing mainly EVOO lignans ( middle ) and fractions containing mainly EVOO secoiridoids ( right ) for 4 days. Cell proliferation, measured using a crystal violet assay as described in "Materials and methods", was expressed as % of untreated cells ( dashed line = 100% cell proliferation). Results are means ( columns ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate. Statistically significant differences (one-factor ANOVA analysis) between HER2-negative MCF-7/pBABE control cells and HER2-positive MCF-7/HER2 cells are shown. All statistical tests were two-sided.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Incubation, Crystal Violet Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Effects of HER2 siRNA on EVOO polyphenols-induced cytotoxicity . ~5 × 10 3 cells (96-well plates) overnight serum-starved SKBR3 ( left ) and MCF-7/HER2 ( right ) cells were transfected with HER2 siRNA or control siRNA (80 pmols/well) before exposure to 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of SKBR3 and MCF-7/HER2 cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means ( dots ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Transfection, Viability Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Influence of EVOO phenolics on the levels of HER2 oncoprotein in SKBR3 breast cancer cells . Overnight serum-starved SKBR3 cells were cultured in DMEM medium-0.1% FBS in the absence or presence of increasing concentrations of EVOO phenolics for 48 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates from EVOO polyphenols-treated and untreated control cells. Results are means ( columns ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells are shown (one-factor ANOVA analysis). All statistical tests were two-sided.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Influence of EVOO phenolics on the levels of HER2 oncoprotein in MCF-7/HER2 breast cancer cells . Overnight serum-starved MCF-7/HER2 cells were cultured in DMEM medium-0.1% FBS in the absence or presence of increasing concentrations of EVOO phenolics for 48 h. The Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates from EVOO polyphenols-treated and untreated control cells. Results are means ( columns ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells are shown (one-factor ANOVA analysis). All statistical tests were two-sided.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Influence of the activation (phosphorylation) status of HER2 tyrosine kinase activity on the cytotoxic activity of EVOO polyphenols . ~5 × 10 3 cells (96-well plates) overnight serum-starved SKBR3 ( left ) and MCF-7/HER2 ( right ) cells were treated with 0.1 μM lapatinib before exposure to 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of treated and untreated control cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means ( dots ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Activation Assay, Activity Assay, Viability Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Influence of EVOO polyphenols on the levels of HER2 tyrosine kinase activity . Overnight serum-starved SKBR3 ( left ) and MCF-7/HER2 ( right ) cells were treated with 100 μM 1-[+]-acetoxypinoresinol and 100 μM DAOA for 0, 6, 24 and 48 hr. Oncogene Science HER2 microtiter ELISA was used according to the manufacturer's instructions to compare HER2 protein concentrations in whole cell lysates throughout the time-course. HER2 expression at the 0 hr-time point was set as 100%. Results are means ( columns ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate. Statistically significant differences between experimental conditions and unsupplemented control cells at the 0 hr-time point are shown (one-factor ANOVA analysis). All statistical tests were two-sided. EVOO polyphenols-treated and untreated control cells (0 hr) were tested in parallel for p185 HER2 autophosphorylation at Tyr1248 using immunoblotting procedures as described in "Materials and methods".

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: Synergy analysis of the concurrent combination with EVOO polyphenols and the anti-HER2 monoclonal antibody trastuzumab in SKBR3 breast cancer cells . ~5 × 10 3 cells (96-well plates) overnight serum-starved SKBR3 cells were treated with 100 μg/ml trastuzumab in the absence or presence of 25 μM 1-[+]-acetoxypinoresinol or 25 μM DAOA. The metabolic status of treated and untreated control cells was evaluated using a MTT-based cell viability assay and Interaction Indexes were calculated by dividing expected cytotoxicities (additive models: sum of cell toxicities induced by each agent alone) by those obtained experimentally in the actual combination (Interaction Indexes < 1, = 1, > 1 < 2, and > 2 denote a nature of the interaction synergistic, additive, antagonistic and protective, respectively). Results are means ( dots ) and 95% confidence intervals ( bars ) of three independent experiments made in triplicate.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques: Viability Assay

Journal: BMC Cancer

Article Title: tabAnti-HER2 ( erb B-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

doi: 10.1186/1471-2407-8-377

Figure Lengend Snippet: EVOO phenolics, HER2, and breast cancer progression: Therapeutic opportunities . Although an appropriate dietary intervention reproducing the prominent anti-HER2 features of EVOO polyphenolsmust be carried out in human pilot studies in the future, it is reasonable to suggest that parental and/or semi-synthetic derivatives of EVOO lignans and secoiridoids might represent previously unrecognized diet-based therapeutic strategies capable to successfully prevent and/or treat HER2-driven natural history of breast cancer disease.

Article Snippet: Testing for the phosphorylation (activation) status of HER2 was performed by immunoblotting procedures using the monoclonal c- erb B2/HER2 (phosphor-specific) antibody Ab-18 (NeoMarkers, Fremont, CA, USA).

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

doi: 10.1152/ajpcell.00141.2010

Figure Lengend Snippet: Primers for semiquantitative RT-PCR

Article Snippet: NRG1 solvent (20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, and 100 mM l -arginine, pH 6.5), recombinant human NRG1β [recombinant human glial growth factor 2 (rhGGF2), 100 ng/ml, a gift from Acorda Therapeutics], ErbB2 receptor inhibitor AG825 (1 μM, Calbiochem, San Diego, CA), or a ErbB1/ErbB2/ErbB4 receptor inhibitor (1 nM, catalog no. 324840, Calbiochem) was added in the culture medium at different time points.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

doi: 10.1152/ajpcell.00141.2010

Figure Lengend Snippet: Expression of neuregulin-1 (NRG1) and ErbB receptors during hanging drop-induced murine ESC differentiation. A: real-time PCR assessment of mRNA expression of NRG1α and NRG1β during ESC differentiation. 18S rRNA was used as loading control. The results are from three independent experiments. B: mRNA expression of ErbB receptors during ESC differentiation. C: protein expression of ErbB receptors during ESC differentiation. D: phosphorylation levels of ErbB receptors during ESC differentiation. mRNA expression of ErbB1, ErbB2, ErbB3, and ErbB4 was measured by semiquantitative RT-PCR. The phosphorylation and total ErbB receptor levels were measured by Western blot analysis. GAPDH was used as loading control.

Article Snippet: NRG1 solvent (20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, and 100 mM l -arginine, pH 6.5), recombinant human NRG1β [recombinant human glial growth factor 2 (rhGGF2), 100 ng/ml, a gift from Acorda Therapeutics], ErbB2 receptor inhibitor AG825 (1 μM, Calbiochem, San Diego, CA), or a ErbB1/ErbB2/ErbB4 receptor inhibitor (1 nM, catalog no. 324840, Calbiochem) was added in the culture medium at different time points.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

doi: 10.1152/ajpcell.00141.2010

Figure Lengend Snippet: Inhibition of ErbB2 or ErbB4 receptors inhibited hanging drop-induced cardiac differentiation of murine ESCs. A: inhibition of ErbB2 abolished NRG1-induced ErbB2 activation in ESCs. Six days after the initiation of hanging drop-induced ESC differentiation, cells were treated with NRG1 (100 ng/ml) for 2 h. An ErbB2 inhibitor (AG825, 1 μM) was added 1 h before NRG1 treatment. Total and phosphorylated ErbB receptor levels were measured by Western blot analysis. Densitometric quantification is shown at right. B: ErbB1/ErbB2/ErbB4 inhibitor abolished NRG1-induced activations of ErbB2 and ErbB4. Cells were treated and total and phosphorylated ErbB receptor levels were measured as described in A. Densitometric quantification is shown at right. C: inhibition of the ErbB2 and/or ErbB4 receptor decreased the protein level of NKX2.5 and the phosphorylation of Akt and ERK1/2. Cells were incubated with AG825 (1 μM) or an ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7. Cells were then incubated without the inhibitor and collected on day 9. NKX2.5, pAkt, and pERK1/2 were measured by Western blot analysis. GAPDH was used as loading control. D: inhibition of the ErbB2 and/or ErbB4 receptor decreased the mRNA of NKX2.5 and cTNT. mRNA expression of NKX2.5 and cTNT was assessed by real-time PCR. The results are from three independent experiments. *P < 0.05 vs. control. E: inhibition of the ErbB2 and/or ErbB4 receptor decreased the percentage of EBs that contained beating areas. The percentage of EBs containing beating areas was measured at each indicated time point. In total, 100 EBs were counted in each experiment. The results are from three independent experiments. *P < 0.05 vs. control.

Article Snippet: NRG1 solvent (20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, and 100 mM l -arginine, pH 6.5), recombinant human NRG1β [recombinant human glial growth factor 2 (rhGGF2), 100 ng/ml, a gift from Acorda Therapeutics], ErbB2 receptor inhibitor AG825 (1 μM, Calbiochem, San Diego, CA), or a ErbB1/ErbB2/ErbB4 receptor inhibitor (1 nM, catalog no. 324840, Calbiochem) was added in the culture medium at different time points.

Techniques: Inhibition, Activation Assay, Western Blot, Incubation, Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: Improving murine embryonic stem cell differentiation into cardiomyocytes with neuregulin-1: differential expression of microRNA

doi: 10.1152/ajpcell.00141.2010

Figure Lengend Snippet: A: microRNA analysis of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was differentially regulated by NRG1 and ErbB receptor inhibition. Cardiac differentiation of ESCs was performed by the hanging drop method. Cells were incubated with NRG1, ErbB2 inhibitor AG825 (1 μM), or ErbB1/ErbB2/ErbB4 inhibitor (1 nM) during day 5–7. Cells were then incubated without stimulation. RNA was collected on day 9. Accession number and name of the microRNAs are shown. B: mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p were differentially expressed during hanging drop-induced mesoderm formation of ESCs. The expression of mmu-miR-296–3p and mmu-miR-200c* was increased during hanging drop-induced ESC differentiation. Expression of mmu-miR- 465b-5p was decreased during hanging drop-induced ESC differentiation. ESC differentiation was induced by the hanging drop method. RNA was collected on days 0, 3, 5, and 9. Expression of mmu-miR-296–3p, mmu-miR-200c*, and mmu-miR-465b-5p was assessed by real-time PCR. Data were normalized to snoRNA202 and are presented as fold expression relative to the mRNA level of undifferentiated ESCs (day 0). The results are from three independent experiments. *P < 0.05 vs. D0; **P < 0.01 vs. D0. C: expression of microRNAs was inhibited by anti-miR inhibitors. ES-D3 cells were transfected with anti-miR inhibitors or scrambled negative control and then the differentiation of ESCs was induced by the hanging drop method. On day 3 of ESC differentiation, RNA was collected and the expression of microRNAs was measured by real-time PCR. Data were normalized to snoRNA202 and are presented as a percentage of microRNA vs. control expression. D: expression of brachyury and NKX2.5 in differentiated ESCs that were transfected with anti-miR inhibitors. RNA collected in C was used for assessing the expression of brachyury and NKX2.5 by real-time PCR. Data were normalized to 18S rRNA and are presented as percent expression of individual microRNA vs. control transfection. *P < 0.05 vs. control.

Article Snippet: NRG1 solvent (20 mM sodium acetate, 100 mM sodium sulfate, 1% mannitol, and 100 mM l -arginine, pH 6.5), recombinant human NRG1β [recombinant human glial growth factor 2 (rhGGF2), 100 ng/ml, a gift from Acorda Therapeutics], ErbB2 receptor inhibitor AG825 (1 μM, Calbiochem, San Diego, CA), or a ErbB1/ErbB2/ErbB4 receptor inhibitor (1 nM, catalog no. 324840, Calbiochem) was added in the culture medium at different time points.

Techniques: Inhibition, Incubation, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control