erbb2 receptor inhibitor ag825  (Millipore)

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