Fig. 1.

Autocrine TGF-β signaling is increased in cells expressing mutant HER2. A. Phase contrast (left) and immunofluorescence (right) images of MCF10A/HER2^WT and MCF10A/HER2^YVMA cells cultured to reach confluence. For immunofluorescence staining, cells growing on glass coverslips were fixed and stained using antibodies against E-cadherin (green) and N-cadherin (red). DAPI (blue), nuclear staining. Bar equals 50 μm. B. MCF10A (left) or BEAS2B (right) cells stably expressing HER2^WT, HER2^YVMA or empty vector were grown in complete medium and treated with lapatinib for 16 h or left untreated. Total RNA was extracted and subjected to reverse transcription followed by quantitative PCR for TGF-β1, -β2 and -β3 as described in Materials and Methods. Data were normalized to the MCF10A/vec or BEAS2B/vec control cells. Each data represents the mean ± S.D. of 3 experiments. C. Cells grown on 100-mm dishes (1×10⁶ cells/dish) were incubated for 24 h in serum-free medium. Conditioned medium (CM) was collected and analyzed for total amount of TGF-β1 by ELISA as indicated in Materials and Methods. Data are normalized to pg/ml/10⁶ cells/24 h. Each data represents the mean ± S.D. of 3 experiments. D. Cells grown on 6-well plate were serum-starved for 16 h before lysed. Cell lysates were subjected to immunoblot using indicated antibodies. E. Top: MCF10A/HER2^WT and MCF10A/HER2^YVMA cells were plated in Matrigel in 8-well chambers and allowed to grow in the absence or presence of LY2109761 at the indicated concentrations. The inhibitor was added to the top medium 12 h after cell seeding and replenished every 3 days. Phase contrast images shown were recorded 9 days after the initial seeding of cells. Bottom: 9-day acini were trypsinized and total cell number was determined in a Coulter counter. Each bar graph represents the mean ± S.D. of 4 wells.