Signaling pathway model development and model calibration.

<p>(A) Simplified Signaling Network Model. Two inputs (growth factor and HGF), signaling protein nodes (EGFR, MET, <i>RAS_m</i>, RAS_w, PI3K/AKT, RAF, MEK, ERK, RSK), and one output (cell viability). Of note, <i>RAS_m</i> indicates a mutant <i>RAS</i>, while RAS_w indicates a wild-type RAS. A green line represents a positive relation (stimulation) and red line represents a negative relation (inhibition). (B) pMET (Y1234/5), pMEK, pERK, pAKT (both T308 and S473), and pRSK (T359) expression after different inhibitors (1 μM), MET inhibitor (METi, PHA665752), EGFR inhibitor (EGFRi, Erlotinib), RAF inhibitor (RAFi, LY3009120), MEK inhibitor (MEKi, GDC0623), ERK inhibitor (ERKi, SCH772984), and AKT inhibitor (AKTi, MK2208) in both control medium (DMSO) and after 2-hour stimulation-by-HGF (50 ng/mL) condition (DMSO plus HGF). (C) Relative cell viabilities after treatments. Cells were treated inhibitors (1 μM) for 72 hours. Cell viabilities were assessed by CellTiter-Glo assay (Promega). Representative triplicates (± SD) are presented, which showed similar results at least three times. (D) The western blots were quantified using ImageJ and relative changes (log2 scale) are reported. Average values of relative cell viabilities are also reported in log2 scale. All the data are normalized to the treatment-naïve control condition. pAKT (T308) readouts are quantified and used in the model. Of note, we didn’t quantify total protein levels because our primary interest was protein activity (protein phosphorylation). The effects of all inhibitors are modeled by assuming very small activity of a target protein (i.e., 1/16 of control, Methods section), and therefore pMET under METi is set be a very small number (i.e., 1/16 of control) for consistency. (E) Comparison between model predictions (gray box plots) and experimental data (black dots). A log2 fold change of pMET, pMEK, pAKT, pERK, pRSK, and cell viability after treatments of different inhibitors (METi, EGFRi, MEKi, ERKi, AKTi) in both a control medium and HGF-stimulated conditions. RMSE of each protein is following. pMET: 0.03; pMEK: 0.49; pAKT:0.33; pERK: 0.96; pRSK: 0.57; and cell viability: 0.47. The numerical data used in Fig 2 are included in the first sheet <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002930#pbio.2002930.s013" target="_blank">S1 Data</a>. AKT (PKB), protein kinase B; DMSO, Dimethyl sulfoxide (control); EGFR, epidermal growth factor receptor; ERK, extracellular receptor kinase; HGF, hepatocyte growth factor; MEK, mitogen-activated protein kinase kinase; MET (c-MET), tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR); PI3K, phosphoinositide 3-kinase; RAF, rapidly accelerated fibrosarcoma; RAS, rat sarcoma; RAS_m, mutated RAS; RAS_w, wild-type RAS; RMSE, root-mean-squared-error; RSK, ribosomal S6 kinase.