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. 2010 Jul 26;120(8):2684–2698. doi: 10.1172/JCI42118

Figure 9. ErbB2 kinase is required for p61-dependent promigratory and prometastatic function.

Figure 9

(A) A549 cells were pretreated with 400 nM Lapatinib or 250 nM PHA-665752 for 2 hours and allowed to migrate in a Transwell insert in response to 7 nM p61-Sema3E in presence of the same inhibitors. Migrated cells were quantified by staining with crystal violet (see Methods). Data are given as average ± SD of 2 independent experiments. **P < 0.005. Analogous results were obtained by analyzing MDA-MB-435 and HeLa cancer cells (see Supplemental Figure 11, A and B). (B) A549 cells were transduced to establish an autocrine circuit of p61-Sema3E, fluorescence labeled with CFDA-SE, and injected systemically into mice treated with Lapatinib or vehicle (see Methods). Forty-eight hours after injection, metastatic fluorescent cells in the lungs were quantified (as described in Methods). The graph indicates average values ± SD of 5 mice per each experimental group. **P < 0.003. (C) The expression of ErbB2 (or Met tyrosine kinase as control) was knocked down in A549 tumor cells by RNAi (see Methods) and migration was assayed as above in response to 7 nM p61, 1 nM Hrg-β, or 1 nM HGF. Data are given as average ± SD of 2 independent experiments. Statistical significance was calculated relative to mock-treated cells in each group. *P < 0.05. (D) ErbB2-depleted A549 cells and respective controls were transduced to establish an autocrine circuit of p61-Sema3E (see expression analysis). Tumor cells were labeled with CFDA-SE before intravenous injection into nude mice. The graph shows the number of metastatic cells infiltrating the lungs after 48 hours, quantified as in B (average values ± SD of 5 mice per each experimental group). **P < 0.005. (E) Sema3E-induced migration of A549 cells was assayed as above, in the presence of DMSO (vehicle), or 2 μm U73122 (PLCγ), 10 μm PD98059 (“PD,” MAPK inhibitor), or 10 μm LY294002 (“LY,” PI3K inhibitor). PLCγ close to U73122; (MAPK inhibitor) close to PD98059, and (AKT inhibitor) close to LY294002. Data are given as average ± SD of 2 independent experiments. *P < 0.05, **P < 0.003.