Fig. 1.

Tet-inducible expression of RON in T47D cells allows dissection of ligand-dependent and ligand-independent signaling. a Titration of RON expression by doxycycline in T47D cells. T47D-RON cells were treated with increasing concentrations of doxycycline for 48 h. Robust ligand-independent activation of RON is detected at higher concentrations of doxycycline, as reflected by increased pRON. b Dose-response stimulation of RON with MSP. T47D-RON cells were treated with doxycycline (100 ng/ml) for 48 h, and cultured in serum-starved media for 24 h. Cells were then stimulated with increasing concentrations of MSP for 15 min. Lysates were immune-precipitated with anti-4G10 phospho-tyrosine antibody, and subjected to Western blot analysis. c Whole cell lysates (WCL) of the same samples were analyzed for pAKT and pERK as readouts for activation of PI3K and MAPK pathways. β-actin was used as loading control. d Western blot shows the efficacy of a small molecule RON inhibitor, ASLAN002, at various concentrations on decreasing ligand-independent RON phosphorylation. Mock-infected T47D cells were used as a negative control. e Effect of ASLAN002 on two types of RON activation: MSP-dependent vs MSP-independent. T47D-RON cells were treated with low-level doxycycline (50 ng/ml) for 48 h. Cells were then serum starved for 24 h and treated with either RON inhibitor (ASLAN002, 1 μM) alone for 4 h, or RON inhibitor followed by MSP stimulation for 30 min. Cell lysates were tested for inhibition of RON and downstream signaling pathways (left panel). For ligand-independent RON activation, T47D-RON cells were treated with high dose of doxycycline (500 ng/ml) for 48 h in normal medium. Cells were then treated with RON inhibitor ASLAN002 (1 μM) for 4 h and analyzed for downstream signaling activity (right panel). GAPDH was used as loading control. Note that the left and right panels are from the same gel, separated for clarity. See also Supplementary Figure S1 and S2. NS non-specific band