Fig. 4

Combinations of 4A7 and gefitinib potently inactivate HER3, EGFR, and their downstream signaling, and markedly induce growth inhibition and cell death in TNBC cells. A, The indicated TNBC cell lines cultured at normal condition were collected and subjected to western blot analyses of EGFR, p-EGFR (Y1068), HER3, and p-HER3 (Y1289). β-actin was used as a loading control. The relative signal intensity obtained via densitometry analyses was shown underneath of each image. B, Equal amount of total protein extracts of MDA-MB-468 (MDA-468) or HCC1806 cells were subjected to IP using a control IgG, anti-EGFR Ab (left), or anti-HER3 Ab (right), and followed by western blot analyses of HER3 and EGFR. C, MDA-MB-468 or HCC1806 cells at normal culture condition were treated with vehicle, 4A7, gefitinib, or the combinations of 4A7 and gefitinib for 24 h. The cells were collected and subjected to western blot assays of the indicated markers. Densitometry analyses of the signals were shown underneath, and the arbitrary numbers indicated the intensities of each image relative to the untreated controls, defined as 1.0. D, MDA-MB-468 or HCC1806 cells were plated in 96-well plates. After 24 h, the culture medium was replaced with fresh medium containing vehicle control (DMSO), gefitinib (4µmol/L) alone, or gefitinib (4µmol/L) plus 4A7 (20 µg/ml) (Gefi + 4A7), and incubated for additional 72 h. The percentages of surviving cells relative to controls, defined as 100% survival, were determined by cell growth/viability (MTS) assays. Data shows a representative of three independent experiments. Bars, SD. *, p < 0.05, **, p < 0.01. E, MDA-MB-468 or HCC1806 cells seeded in 6-well plates were treated with gefitinib (8µmol/L) or its combinations with 4A7 (20 µg/ml) for 48 h. The cells were subjected to the LIVE/DEAD Cell Imaging assays. Green, live cells; red, dead cells. The ratio of dead/live cells was shown by in each sample. Data shows a representative of three independent experiments