To measure pathway activity, we linearly combined the logarithmic expression values of the genes in each pathway using a weight of −1 for repressors to account for the accumulative effect of all the genes in a pathway.
In order to identify mutated genes beyond KRAS, BRAF and NRAS that might account for expanded RAS pathway activity, we stratified these 468 CRCs using an 18-gene RAS pathway gene expression signature score that measures pathway activation via MEK functional output16.
We believe this reflects the complexity of these cell signaling pathways combined with the power of the signature development approach whereby the capacity to sample a diverse array of expression values can yield measures of pathway activity.
Each data set was scored against network models representing different aspects of inflammatory signaling and cell cycle progression, and these scores were compared with independent measures of pathway activity in NHBE cells to verify the approach.
In order to measure the pathway activity, we first measured Ras protein activity levels able to stimulate the ERK cascade.
Also, Siglecs exhibit distinct binding specificities, and different organisms might express different patterns of sialylated ligands; this aspect should be taken into account when measuring Siglec pathway activity.
Luciferase assay measuring Wnt/βcatenin pathway activity 20 hr after over-expression of BATLuc and Renilla constructs together with control, T, Hoxa13, or Hoxa13+T.
Importantly, the Wnt1-expressing T47D cells have an approximately three-fold increase in active β-catenin levels compared with control cells, attesting to the ability of the antiserum to measure canonical pathway activity.
(D – E ) Luciferase assay measuring Wnt/βcatenin pathway activity after over-expression of the BATLuc construct together with a Renilla-expressing vector and either (D ) control, Hoxa9, Hoxa13 or the combination of Hoxa9 and Hoxa13 expressing vectors.
Instead, most of the genes in these signatures reflect downstream transcriptional consequences of the perturbation, which may nevertheless provide better measures of upstream pathway activity [ 5].
