Training Signaling Pathway Maps to Biochemical Data with Constrained Fuzzy Logic: Quantitative Analysis of Liver Cell Responses to Inflammatory Stimuli
Figure 6
Validation of cFL crosstalk predictions.
(a) Analysis of systematic error as well as the topologies of the family of trained cFL models (Figure 5) indicated that c-Jun was partially activated after TGFα stimulation. Models with crosstalk from Ras or PI3K to Map3K1 predicted that JNK was partially activated under these experimental conditions even though it was not partially activated in the dataset. We tested whether JNK was actually partially activated under these conditions by stimulating HepG2 cells with TGFα and measuring levels of phosphorylated JNK and c-Jun by a bead-based antibody assay after 30 minutes. Fold increase in measured phosphorylation over un-stimulated control for c-Jun (black) and JNK (red) is shown. Where available, biological replicates are indicated with filled circles. Solid lines indicate the averages of the replicates. This experiment indicates that JNK was partially phosphorylated under TGFα stimulation and the cFL models with crosstalk from Ras or PI3K to MAP3K1 were correct. (b) CFL analysis of the topologies and fit of the HepG2 training dataset to several PKNs suggested that IL6 activated downstream nodes through the Ras/MEK pathway (Table 3). To test this prediction, a validation dataset was examined [36]. This validation dataset showed that the activation of nodes other than STAT3 that responded robustly to IL6 stimulation was ablated by pretreatment with a small molecule MEK inhibitor but not other inhibitors, demonstrating that the Ras/Raf/MEK pathway mediates this crosstalk.