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. 2008 Jul 28;182(2):381–393. doi: 10.1083/jcb.200712066

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© 2008 Ghosh et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 8. — The Gαi3–GIV signaling complex is necessary for macrophage chemotaxis and tumor cell migration. (A) GIV, but not Gαi3, expression is induced ∼18-fold upon differentiation of monocytes into macrophages. THP1 monocytes were treated with 5 nM TPA or DMSO carrier, and the expression of Gαi3 and GIV was followed by immunoblotting over 68 h. (B) Depletion of Gαi3 or GIV impairs macrophage chemotaxis. (Top) Schematic illustration of the chemotaxis assay in which macrophages migrate toward a pipette tip, continuously releasing fMLP to maintain a gradient. The dashed line denotes the edge of the monolayer just before the application of the chemotactic gradient. (Bottom) THP1-derived macrophages treated with scr (a and b), Gαi3 (c and d), or GIV (e and f) siRNA were imaged at the end of a fMLP chemotaxis assay. Arrow denotes direction of chemotaxis. Bars: (a, c, and e) 250 μm; (b, d, and f) 50 μm. (C) Immunoblotting performed on lysates harvested at the end of the chemotaxis assay (shown in B) demonstrating that depletion of Gαi3 or GIV impairs Akt activation (pAkt) in THP1-derived macrophages. (D) GIV is expressed in colon cancer cell lines with high (HCT-116, DLD1) but not low (HT-29p, LS-174T) in vivo metastatic potential. Gαi3 and GAIP (used as a control) expression are similar in all cell lines. (E) Depletion of Gαi3 or GIV impairs wound healing in highly metastatic colon cancer cells. DLD1 cells treated with scr (a and b), Gαi3 (c and d), or GIV (e and f) siRNA were monitored at 0 and 36 h after scratch-wounding. Bars, 1 mm. (F) Depletion of Gαi3 (left) or GIV (right) impairs Akt activation (pAkt) in highly metastatic DLD1 colon cancer cells. Immunoblots were performed on lysates harvested at the end of a wounding assay.