Fig. 2.

Autocrine TGFβ signaling regulates the protrusive, mesenchymal phenotype of EMT6 cells via the Par6 pathway. (A and B) EMT6 cells were grown in Matrigel for 5 days and continuously treated with an anti-TGFβ1 Ab (αTGFβ1) at 10 μg/mL or left untreated, as indicated. Bright field images of 3D structures are shown in A, while quantification of protrusive structures formed in control or αTGFβ1-treated cultures is shown in B. (C) Characterization of a Par6S345P (pPar6) Ab in NMuMG cells. Lysates from parental cells or cells expressing Flag-tagged wt or Par6/S345A were subjected to IP with Abs to Par6 or Flag and immunoblotted for pPar6. A pPar6 band was readily detected in wt but not the Par6/S345A IPs. Endogenous Par6 was not detected in NMuMG parental (P) cells after total Par6 IP (Left), but co-precipitated with TGFβ receptor I (TGFβRI), in which case TGFβ1 (500 pM, 1.5 h) stimulated Par6 phosphorylation. In Par6/wt overexpressing NMuMG, elevated levels of pPar6 were detected. (D) Analysis of endogenous pPar6 in EMT6 cells. Lysates from EMT6 cells subjected to irrelevant Ab IP (Ir Ab) or a Par6 IP were then blotted for pPar6. pPar6 that was present in untreated cells was enhanced by TGFβ1 treatment and was reduced by neutralizing TGFβ Ab (αTGFβ1), but not by 10 μM of the type I kinase inhibitor SB431542 (SB). Lysates were also blotted for pSmad2, which revealed inhibition of autocrine activation by both the neutralizing Ab and SB431542. In the Right, phosphorylation of Par6 in wt or Par6/S345A-expressing cells was analyzed by immunoblotting. (E) Bright field images of EMT-6 pools expressing empty vector, Par6/wt, or Par6/S345A grown in Matrigel. Quantification of the percent of structures with protrusive morphology (mean +/− SD from three independent experiments shown at the bottom of each image; see SI Text for details) shows that Par6/S345A expression significantly suppresses (P < 0.005) the percent of protrusive structures formed by EMT-6 cells. (Scale bar in A, 50 μm; E, 100 μm.)