ECM invasion by co-cultures of squamous cell carcinoma and cancer-associated stromal fibroblasts

Based on the suggestion that MEK activation is reduced in combination with increased ERK Darapladib dephosphorylation 1357470-29-1 cost activity in MG132-treated cells, we sought to parse these two effects quantitatively. To accomplish this, we devised a kinetic modeling scheme. Given the potentially complex effects of MG132 treatment on growth factor receptor-mediated signaling upstream of ERK1/2, our strategy was to fit each MEK1/2 phosphorylation time course to an empirical function, which serves then as the input to a modified Michaelis-Menten model of ERK phosphorylation and dephosphorylation on its two activating sites. In the case of the time courses with MG132, this model tests the consistency of the simplest hypothesis: that the phosphatase activity of the enzyme catalyzing dephosphorylation of the two sites on ERK1/2) is enhanced by a constant factor, while the rest of the parameters affecting ERK phosphorylation kinetics were constrained to have the same values in MG132- treated and control cells. This model was iteratively fit to the ERK data set by Monte Carlo sampling of the model parameters to obtain a large ensemble of parameter sets that produce nearly equivalent qualities of fit, allowing us to evaluate the degree to which each parameter was properly constrained. As a central estimate of the model output, the mean of the ensemble is quantitatively consistent with the corresponding ERK phosphorylation data. The corresponding estimate of the fold-upregulation of ERK phosphatase activity in MG132-treated cells is 3.6160.15. The small coefficient of variation indicates that this parameter was tightly constrained by the data. Our computational analysis supports a hypothetical model whereby MG132 treatment reduces ERK phosphorylation by both reducing MEK activation and enhancing ERK dephosphorylation. Hence, we sought to confirm that DUSPs implicated in ERK1/2 dephosphorylation, such as DUSP1/MKP1 and especially DUSP6/MKP3, are upregulated in our MG132-treated cells. The effects of MG132 on basal and growth factor-modulated levels of DUSP expression were found to depend on the treatment time, consistent with the time scale of protein synthesis and turnover. As reported previously, a 30-minute pretreatment with MG132 was insufficient to alter the