Figure 5.

HER2 overexpression enhances transcriptional induction of an angiogenesis/hypoxia module upon treatment with EGF. (a) Total RNA was subjected to analysis of the indicated genes by PCR, as in Figure 4a. (b) Schematic presentation of the angiogenesis module. Narrow arrows indicate transcriptional upregulation. AREG, amphiregulin. (c) Immunoblot analysis was performed on HER2-overexpressing acini treated without or with EGF for the indicated time intervals, using hypoxia-inducible transcription factor-2α (HIF2α) and LOXL2 antibodies. (d, e) MCF10A/HER2 cells were plated in Matrigel and grown for 4 days in the presence of EGF. Thereafter, the cultures were grown for 8 additional days in the absence or presence of the indicated concentrations of a lysyl oxidase inhibitor, β-aminopropionitrile (BAPN). Capturing of photos and morphometric analyses of 50 acini were performed 8 days later. (f) MCF10A/HER2 cells were plated in Matrigel in the absence or presence of a recombinant LOXL2 enzyme (1 μm), and then incubated for 18 days, before phase microscopy (top) and staining with 4,6-diamidino-2-phenylindole (DAPI) and an anti-Laminin antibody (bottom; bars, 100 μm). White arrows mark outgrowths across the Laminin shell.