Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HER2 missense mutations have distinct effects on oncogenic signaling and migration.

doi: 10.1073/pnas.1516853112

Figure Lengend Snippet: Fig. 2. HER2 V777L mutation increases signaling pathway activation but does not promote transformed phenotypes in MCF-10A cells. (A) MCF-10A HER2 mutant and control cell lines were grown in physiologic (0.2 ng/mL) EGF supplemented assay media and subjected to Western blotting analysis with the indicated antibodies. White lines represent lanes that have been removed from blot. (B) Relative mean (±SEM) proliferation data for V777L clones and MCF- 10A controls in 0.2 ng/mL EGF supplemented assay media (n ≥6 per cell line, two independent experiments were performed). (C) Representative images depicting soft agar colony formation for MCF-10A V777L, controls, and an MCF-10A cell line overexpressing the HER2 V777L cDNA. (Scale bar, 200 μm.) (D) 3D acinar morphogenesis assay for MCF-10A HER2 mutant isogenic cell lines and mutant HER2 overexpression cell lines. (Scale bar, 50 μm.)

Article Snippet: A wild-type HER3 expression vector was created by subcloning HER3 cDNA from the pCMV6-XL4-ERBB3 plasmid (SC118918; Origene) into a pIRES-neo3 backbone.

Techniques: Mutagenesis, Activation Assay, Transformation Assay, Control, Western Blot, Clone Assay, Over Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HER2 missense mutations have distinct effects on oncogenic signaling and migration.

doi: 10.1073/pnas.1516853112

Figure Lengend Snippet: Fig. 6. Cells harboring both HER2 V777L and PIK3CA E54K mutations have increased migratory capacity in vitro and increased interaction between HER3 and p85. (A) Relative fraction of scratch wound closure (±SEM) as measured after 16 h. Cells were grown to near confluent monolayers, a wound was introduced, and assay media without EGF ± lapatinib (1 μM) was added back to wells (n ≥14, at least three independent experiments were performed, ***P ≤0.001, one- way ANOVA followed by Bonferroni multiple comparison test). (B) Representative phase contrast images of wound closure. (C) Relative fraction of scratch wound closure (±SEM) for MCF7 and HER2 mutant derivatives in serum-supplemented media ± lapatinib (5 μM) after 20 h (n ≥12, *P ≤0.05, at least two independent experiments were performed, one-way ANOVA followed by Bonferroni multiple comparison test). (D) Representative images from MCF7 cell line scratch wound closure experiments. (Scale bar, 500 μm in B and D.) Mean (±SEM) cell velocity (Left) and persistence (net cell displacement to total distance traveled ratio) (Right) in microchannel migration experiments of MCF-10A + E545K and V777L double knockin cell lines (E) and MCF7 and MCF7 V777L cell lines (F) (n ≥30 tracked cells, at least three independent experiments were performed, ***P ≤0.001, **P ≤0.01, *P ≤0.05, unpaired t test).

Article Snippet: A wild-type HER3 expression vector was created by subcloning HER3 cDNA from the pCMV6-XL4-ERBB3 plasmid (SC118918; Origene) into a pIRES-neo3 backbone.

Techniques: In Vitro, Comparison, Mutagenesis, Migration, Knock-In

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HER2 missense mutations have distinct effects on oncogenic signaling and migration.

doi: 10.1073/pnas.1516853112

Figure Lengend Snippet: Fig. 7. HER2 mutations do not enhance tumor growth or invasion in vivo. (A) Mean (±SEM) in vivo tumor growth of MCF7 HER2 mutants in mice supplemented with estrogen pellets (n ≥5 animals per group, two independent experiments were performed). (B) Mean (±SEM) in vivo tumor growth for MCF7 HER2 mutants in mice without estrogen pellet supplementation. n ≥5 animals per group. (C) MCF-10A + E545K, L755S, and V777L DKI cells do not form tumors as xenografts in nude mice. (D) Representative images of H&E stained lung sections from tail vein injection assays showing benign lung parenchyma and lack of proliferating, multicellular sites of carcinoma. n ≥5 animals per group. (Scale bar, 100 μm.) (E) MCF7 parental, MCF7-corrected, and HER2 mutant cell lysates were immunoprecipitated with a HER3 antibody. Antibody pulldowns were then subjected to Western blotting analysis with the indicated primary antibodies. One percent of total protein was used as a control (Left). Bar graphs represent the ratio of p85 after immu- noprecipitation to HER3 input control Western blot band as measured with ImageJ software (Right). Bars for MCF7 V777L and MCF7 corrected + V777L represent the average of two clones.

Article Snippet: A wild-type HER3 expression vector was created by subcloning HER3 cDNA from the pCMV6-XL4-ERBB3 plasmid (SC118918; Origene) into a pIRES-neo3 backbone.

Techniques: In Vivo, Staining, Injection, Mutagenesis, Immunoprecipitation, Western Blot, Control, Software, Clone Assay