Fig. 3

Signaling through mTORC1 is required for RON-mediated colony formation and migration in T47D-RON cells. a Representative Western blots for analysis of potential kinases upstream of rpS6 using specific inhibitors of pan-RSK (BI-D1870), mTORC1 (Rapamycin), and RON (ASLAN002). Treatment of T47D-RON cells in ligand-independent conditions with various doses of the inhibitors for 4 h shows almost complete inhibition of phospho-rpS6 by rapamycin, which also showed inhibition of p70S6K activity. See also Supplementary Figure S4A for signaling analysis using specific inhibitors of p70S6K. b Effect of Raptor knockdown on phosphorylation of rpS6 in T47D-RON cells. Cell lysates derived from T47D-RON cells stably expressing three different shRNA against Raptor or control shRNA (scramble), in the absence and presence of 500 ng/ml doxycycline, were subjected to immunoblotting. c–e Effect of mTORC1 knockdown on colony formation of T47D-RON cells. T47D-RON cells infected with scramble or Raptor shRNA construct # 3 (see panel b) were seeded at a very low density in the presence and absence of doxycycline and were allowed to form colonies, followed by crystal violet staining. Representative images are shown in panel c, whereas number and average area per colony are shown in panels d and e. Error bars indicate SD, n = 3. *P < 0.05, **P < 0.005, ***P < 0.0005 (one-way ANOVA, multiple comparisons). f, g Wound healing assays were performed to assess the effect of mTORC1 knockdown on the migration of T47D-RON cells. Doxycycline-treated (500 ng/ml) and untreated T47D-RON-shRaptor and T47D-RON-sh-Scramble cells were seeded at high density the day before wounding. The rate of wound closure in each group over the course of treatment is shown in panel f. Representative images at day 6 are shown in panel g. The initial wound in each group is shown in blue/green; migrating cells from the initial wound are shown in blue. Scale bars represent 300 μm. Data are shown as mean ± SEM, n = 7