Figure 4. Up-regulation of AnxA6 in the basal-like HCC1806 breast cancer cells is associated with decreased xenograft tumor growth.

(A–D) Control (1806-EV) and HCC1806 expressing flag-tagged AnxA6 (1806-Anx6) cells were grown until 80% confluent and harvested by scrapping. WCE were prepared as described in materials and methods, and equal amounts of protein were used in GTPase activation assays. A) Typical Ras activation assay in the presence and absence of GTP-γS. B) Analysis of AnxA6 expression (WCE-WB) and pull-down assays for GTP-γS bound Ras (B) or GTP-γS bound Cdc42 (B) using GST-Raf-1 binding domain (RDB) and GST-PAK-1 binding domain (PDB) respectively by Western blot analysis. C and D) Densitometric analysis of GTP bound Ras or GTP bound Cdc42. Bars represent GTP bound Ras (C) or GTP-bound Cdc42 (D) levels relative to the total levels of the respective GTPases in the control 1806-EV and 1806-AnxA6 cells from at least two independent determinations. (E–F) Migration and invasion assays. Serum-starved cells were plated in Boyden chambers with (E) or without (F) a thin layer of Matrigel in serum free medium. Complete medium containing 10% FBS or EGF in SFM were used as chemoattractants. (G) Control (1806-EV) and HCC1806 expressing flag-tagged AnxA6 (1806-Anx6) cells were grown until 70% confluent, then serum starved for 24 h and incubated with EGF 50 ng/ml in HBSS containing 0.5 mM Mg²⁺, and 0.5 mM Ca²⁺ for the indicated time points. Cells were harvested and the activation of ERK1/2 analyzed by western blotting. (H) Control and flag-tagged AnxA6 expressing HCC1806 cells were injected into mammary fat pads of Nu/J mice and the tumor size and weight were determined as described in Figure 3. ^*p < 0.05; ^**p < 0.01.