Journal: PLoS ONE

Article Title: Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with 125 I-EGF

doi: 10.1371/journal.pone.0024739

Figure Lengend Snippet: The binding of 125 I-EGF to cultured A431 cells treated with complete medium (red), serum free medium (blue), 1 µM of lapatinib (A) or gefitinib (B) in complete medium (green) or 1 µM lapatinib (A) or gefitinib (B) in serum free medium (black) was measured in LigandTracer Grey. A) Lapatinib alters the kinetics of the interaction, as shown as a faster overall association rate and dissociation rate. In serum free medium, the difference between the first and second concentration (indicated by an arrow) is slightly less. B) Gefitinib increases the affinity, observed as a slower dissociation rate and small difference in signal height between the first and second concentration (arrow). The effect of gefitinib is larger in serum free medium.

Article Snippet: The tyrosine kinase inhibitors gefitinib and lapatinib was from Biaffin GmbH (Kassel, Germany) and GlaxoSmithKline (London, UK) respectively.

Techniques: Binding Assay, Cell Culture, Concentration Assay

Journal: Journal of Carcinogenesis

Article Title: Continuous requirement of ErbB2 kinase activity for loss of cell polarity and lumen formation in a novel ErbB2/Neu-driven murine cell line model of metastatic breast cancer

doi: 10.4103/1477-3163.90443

Figure Lengend Snippet: Characterization of ErbB2/Neu-driven mouse mammary tumor cell line (MPPS1): (a) Western blots comparing the expression of ErbB2/Neu in primary mammary tumors (PT1, PT2) from MMTV-Neu Tg mouse to the levels at the indicated passages of MPPS1 cell line (derived from PT1). 100 μg of total protein was loaded per lane. GAPDH is used as a loading control. (b, c) MPPS1 cells grown on glass coverslips were fixed and stained with pan-keratin (KRT), vimentin (VIM) and anti-ErbB2 (ErbB2/Neu) antibodies and detected with fluorochrome-conjugated secondary antibodies. The confocal image shows pan-keratin stained in red and vimentin in green; Scale bars in B and C are 20 μm in length. (d) Sensitivity of MPPS1 to growth inhibition by lapatinib - MPPS1 cells plated in 96-well plates as described in the methods section were treated with an increasing concentrations of lapatinib for 72 hours. The % of viable cells was estimated based on MTT assay. The calculated IC50 value (using Graphpad Prizm software) was 0.125 ± 0.001μM. (e) Concentration-dependent decrease in pY-ErbB2 levels in MPPS1 cells following treatment with lapatinib at the indicated concentrations. Cells were treated with the indicated concentrations of lapatinib for 48 hours. The reduction in pY-ErbB2 levels were assessed by SDS-PAGE/Western blotting analysis from equal amount of total protein lysates (100 μg). Hsc70 was used as loading control.

Article Snippet: The ErbB receptor tyrosine kinase inhibitor lapatinib[ ] was from LC laboratories (Woburn, MA) and was stored as a 1mM solution in dimethyl sulfoxide (DMSO).

Techniques: Western Blot, Expressing, Derivative Assay, Control, Staining, Inhibition, MTT Assay, Software, Concentration Assay, SDS Page

Journal: Journal of Carcinogenesis

Article Title: Continuous requirement of ErbB2 kinase activity for loss of cell polarity and lumen formation in a novel ErbB2/Neu-driven murine cell line model of metastatic breast cancer

doi: 10.4103/1477-3163.90443

Figure Lengend Snippet: Analysis of the requirement of ErbB2 kinase activity and downstream signaling pathways in abnormal polarity and associated hyper-proliferation of MPPS1 cells in 3-D culture: (a) Concentration-dependent decrease in pY-ErbB2 in MPPS1LA cells, grown in 3-D Matrigel following treatment with Lapatinib. Cells were treated with the indicated concentrations of Lapatinib for 48 hours. The reduction in pY-ErbB2 levels was assessed by SDS-PAGE/Western blotting analysis from equal amounts of total lysate protein (100 μg). Hsc70 was used as a loading control. (b) Kinetics of decrease in pY-ErbB2 in MPPS1LA cells grown in 3-D. 50 μg total lysate protein was analyzed for each indicated time point. Blot strips were probed first with anti-pY ErbB2, anti-pAKT and anti pERK1/2, and then stripped and reprobed for ERBB2/Neu, AKT and ERK, respectively. (c) MPPS1LA grown on Matrigel for 8 days, were treated with the indicated concentrations of Lapatinib for another 6 days. The changes in colony morphology were recorded as described in the legend to . Representative images are shown to illustrate the concentration-dependent effect of Lapatinib on the morphology and size of 3-D structures.

Article Snippet: The ErbB receptor tyrosine kinase inhibitor lapatinib[ ] was from LC laboratories (Woburn, MA) and was stored as a 1mM solution in dimethyl sulfoxide (DMSO).

Techniques: Activity Assay, Protein-Protein interactions, Concentration Assay, SDS Page, Western Blot, Control

Journal: Journal of Carcinogenesis

Article Title: Continuous requirement of ErbB2 kinase activity for loss of cell polarity and lumen formation in a novel ErbB2/Neu-driven murine cell line model of metastatic breast cancer

doi: 10.4103/1477-3163.90443

Figure Lengend Snippet: Characterization of Lapatinib-induced normalization of epithelial cell polarity in 3-D Matrigel cultures of MPPS1 cell line. (a) MPPS1 LA cells were grown on Matrigel for 5 days and then were treated with 0.1 μM Lapatinib for 3 days, visualized using phase-contrast microscopy and photographed. Shown is a comparison of the morphology for 3-D structures in cultures treated with DMSO (left panel) or 0.1 μM Lapatinib (right panel)., (b-d) Cells grown on Matrigel (similar to 6A) in 8-well chamber slides were treated with DMSO or 0.1 μM Lapatinib. Following treatment, the cells were paraformaldehyde fixed and stained with anti-ZO1 (B) or anti-GM1 (c; apical marker) or anti-E-cadherin (D) followed by Alexa488-conjugated secondary antibody and Alexa594-phalloidin (for actin). The slides were mounted with a medium containing DAPI and imaged; (e) Lapatinib treatment-induced accumulation of E-cadherin protein levels in MPPS1LA cells; shown is a western blot analysis of 25 μg total proteins from lysates of MPPS1LA cells treated with the indicated concentrations of Lapatinib for 24h. Hsc70 is shown as a loading control.

Article Snippet: The ErbB receptor tyrosine kinase inhibitor lapatinib[ ] was from LC laboratories (Woburn, MA) and was stored as a 1mM solution in dimethyl sulfoxide (DMSO).

Techniques: Microscopy, Comparison, Staining, Marker, Western Blot, Control

Journal: Journal of Carcinogenesis

Article Title: Continuous requirement of ErbB2 kinase activity for loss of cell polarity and lumen formation in a novel ErbB2/Neu-driven murine cell line model of metastatic breast cancer

doi: 10.4103/1477-3163.90443

Figure Lengend Snippet: Reversal of Lapatinib-induced normalization of epithelial cell polarization in 3-D Matrigel upon removal of inhibitor: MPPS1LA cell grown in 6-well plates on Matrigel were treated with 0.1 μM Lapatinib or DMSO vehicle for the indicated times. For evaluating the reversibility of Lapatinib-induced normalization of epithelial architecture and lumen formation, control and Lapatinib-treated 3-D structures were harvested from Matrigel cultures, spun, washed with PBS and replated in fresh Matrigel. The cultures were imaged at the indicated times. The results are representative of three experiments. a, Western blotting of 50 μg protein confirming the effects of Lapatinib-treatment on pY-ErbB2 and total ErbB2 levels and a time-dependent recovery of pY-ErbB2 following Lapatinib withdrawal. b, Digital images of cell structures, left untreated, Lapatinib treated (24 h), or recovery (48 or 96 h) after Lapatinib withdrawal, are shown. Scale bars are 100 μm.

Article Snippet: The ErbB receptor tyrosine kinase inhibitor lapatinib[ ] was from LC laboratories (Woburn, MA) and was stored as a 1mM solution in dimethyl sulfoxide (DMSO).

Techniques: Control, Western Blot