We also confirmed that downstream UPR target genes like CHOP and BiP mRNA were up-regulated by ER stress in each of the cell lines tested (Figure S2).
Given the subtlety (most changes were < two-fold) and specificity (only ∼5% (294/5685) of expressed ORFs were significantly modified) of tif4631Δ on global translation (Fig. S6 and Discussion), the biological significance of these effects will need to be confirmed in downstream experiments.
We also confirmed that downstream ER stress response pathways were activated as in the culture systems.
The concentration of each library was determined by measuring absorbance and confirmed for downstream processing if the OD 260/280 was approximately 1.8.
In case that the sample is composed of a mixture of DNA from different species (e.g., from an air sample), the results will hint to a "candidate mold" which should be further confirmed by downstream analysis, such as sequencing.
Western blot analysis showed that, at different time points post radiation, there is a decrease in the phosphorylation levels of mTOR (p-mTOR) in HCT116 FBXW7+/+ cells while there is no change in HCT116 FBXW7−/− cells, which is confirmed by downstream the phosphorylation levels of s6 Ribosomal Protein (p-s6rp) (Fig. 1A).
More detailed biochemical analysis of 10 confirms typical downstream responses of PPARα agonism including PPARα upregulation, induction of target genes, and inhibition of cell migration.
To confirm the downstream signaling of miR-17-5p, we silenced MKP7, FoxO3a, and HIF1 α (Supplementary Figure S4c).
Importantly, the expression levels of classical target genes for Wnt/β-catenin, AXIN2, and Notch signalling, HEY1, were indeed elevated in LDH, confirming active downstream signalling.
To confirm the downstream effects of DKK-1 suppression, the level of active β-catenin was evaluated after the treatment with IL-32γ or Wnt3a (positive control).
